[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/Developmental Toxicity Screening Test (OECD 421, GLP, Key, rel.1): NOAEL(fertility and developmental) = 1000 mg/kg bw/day in rats. No adverse effect reported at the highest dose level tested in this study conducted with AA 15.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29-Jul-2009 To 04-Mar-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanRcc: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: (P) 11 weeks
- Weight at study initiation: (P) Males: 298 - 342 g; Females: 187 - 207 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no. 15/09) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: 7 days minimum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): range: 22 ± 3 °C
- Humidity (%): range: 30 - 70%)
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: 29-Jul-2009 To: 28-Sep-2009

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle : had been prooved as appriopriate in previous studies
- Concentration in vehicle: 200 g/ml
- Amount of vehicle: dose volume = 5 mL/kg body weight
- Lot/batch no.: 130022563006152

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor. The stability of the test item formulations was considered to be sufficient for weekly preparation based on the dose formulation analyses performed for non GLP study: Dose Range-Finding for Reproduction/ Developmental Toxicity Screening Test in the Wistar Rat. Stability analyses were repeated for this study.
The substance was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Test item formulation was stirred at least 15 minutes until a homogenous solution was prepared. Afterwards test item formulation was stirred very gently or incubated at room temperature without steering until discarding any foam.
Homogeneity of the test item in the vehicle was maintained during the daily administration periodusing a magnetic stirrer.
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male of the same group which had already mated successfully. The reproductive organs of infertile females were examined histopathologically in order to ascertain the reason for the infertility.
- After successful mating each pregnant female was caged : 9/10 females were pregnant in groups 1 (contrl) and 2 (100 mg/kg bw/day) and 10/10 in groups 3 (300 mg/kg bw/day) and 4 (1000 mg/kg bw/day)
- All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day sample from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an evaporative light scattering detector (ELSD) following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The substance peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of AA 15 and, therefore, it was confirmed that only PEG 300 was administered in the control part of the experiment.

The application formulations investigated during the study were found to comprise
The substance in the range of 95.7% to 104.5% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the substance in the preparations was approved because single results found did not deviate more than 3.4% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept 7 days at room temperature due to recoveries in the range of 2.6% to 6.1% which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and PEG 300 as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Males: 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Details on study schedule:

Acclimatization: 7 days minimum (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days (males); 14 days maximum (females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After 28 days of treatment (males); on day 4 post partum (females)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

actual ingested

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

actual ingested

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : see attached document

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males (weekly during pre-pairing period); females (pre-pairing period; days 1 - 8 and 8 - 14 of the pre-pairing period; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum). No food consumption was recorded during the pairing period.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

SACRIFICE

Males were sacrificed after they had been treated for 28 days. Dams were sacrificed on day 4 post partum.

Females which did not give birth on the expected date (day 21 post coitum) were sacrificed on day 25 post coitum.


NECROPSY

All parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy.

For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.


ORGAN WEIGHTS

The testes and epididymides of all parental males were weighed as pairs.


TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.


HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS–hematoxylin.


HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator (histopathology phase). The same applied to all occurring gross lesions and to the female which had to be terminated for ethical reason.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.

Postmortem examinations (offspring):

SACRIFICE

Pups were sacrificed on day 4 post partum.


NECROPSY

All pups were sacrificed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pushing head through the bedding material and salivation in both sexes at 300 and 1000 mg/kg bw/day; diarrhea in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day; activity reduced for 1-3 days in males at 1000 mg/kg bw/day.

CONCLUSION: No adverse effects

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Except for 1 female (no. 66) at the dose level of 300 mg/kg bw/day, all animals survived until the scheduled necropsy.

CONCLUSION: No adverse effects

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reversible and not adverse reduction of body weights and body weight gain in males at 300 and 1000 and in females at 1000 mg/kg bw/day.

CONCLUSION: No adverse effects

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reversible and not adverse reduction of food consumption in males at 1000 and in females at 300 and 1000 mg/kg bw/day

CONCLUSION: No adverse effects

Ophthalmological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

1 IN-LIVE DATA - PARENTAL ANIMALS

1.1 CLINICAL SIGNS OR OBSERVATIONS

See attached tables on pp. 1-3

Except for 1 female (no. 66) at the dose level of 300 mg/kg bw/day, all animals survived until the scheduled necropsy.

In female no. 66, rales, ruffled fur and rhinorrhea were observed on days 2 and 3 of the lactation period. Because these symptoms were accompanied by a significant body weight loss (it lost 8 and 10% of body weight on days 2 and 3, respectively) an application error was assumed and this female was killed for ethical reason on day 3 of the lactation period.

No clinical signs were observed in the control group and at the dose level of 100 mg/kg bw/day.

At the dose levels of 300 and 1000 mg/kg bw/day, pushing head through the bedding material and salivation after application were observed in all males and females at both dose levels during the most of the treatment period. These clinical signs were considered not to be adverse but a result of discomfort caused by the test item administration.

During the pre-pairing period a slight diarrhea was observed in 3 males (nos. 23, 24 and 30) at the dose level of 300 mg/kg bw/day and in 6 males (nos. 31, 33, 36, 37, 38 and 39) and 2 females (nos. 72 and 79) at the dose level of 1000 mg/kg bw/day.
In addition, a reduced activity was observed in 7 males (nos. 31, 32, 33, 34, 35, 37 and 39) at the dose level of 1000 mg/kg bw/day.
Both these effects were considered to be test item-related but not adverse since in all affected animals they were transient (occurred only for a period of 1 to 3 days) and had no observable influence on the wellbeing of the animals.

Because of isolated occurrence following symptoms: rales in 2 males (nos. 35 and 37) at the dose level of 1000 mg/kg bw/day observed for 1 day during pre-pairing period and a watery discharge in 1 female (no. 73) at the dose level of 1000 mg/kg bw/day observed on days 8 and 9 of the pre-pairing period were considered not to be test item-related.


1.2 FOOD CONSUMPTION OF MALES

See attached tables on pp. 4, 8

Pre-pairing Period

No effects on mean food consumption were observed at the dose levels of 100 and 300 mg/kg bw/day.

Treatment of males with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption. From day 1 to 8 of the pre-pairing period, mean food consumption at the high dose was 15.5 compared to 20.5 g/animal/day in the control group. After this period mean food consumption recovered and was similar to the respective control value. Therefore the reduction of food consumption was considered not to be adverse.

The overall differences in mean food consumption of males during the pre-pairing period were: +2.3%, ±0.0% and -13.0% at the dose levels of 100, 300 and 1000 mg/kg bw/day respectively (percentages refer to the respective value in the control group).


1.3 FOOD CONSUMPTION OF FEMALES

See attached tables on pp. 5-7; 9-11

Pre-pairing, Gestation and Lactation Periods

No effect on mean food consumption was observed at the dose level of 100 mg/kg bw/day during the entire treatment period.

Treatment of females with the test item at the dose level of 300 mg/kg bw/day resulted in mean food consumption slightly lower than the respective control values from day 1 to 8 of the pre-pairing period. After this period mean food consumption increased and was similar to the respective control values during the second week of the pre-pairing period as well as during the gestation period. Therefore the reduction of food consumption was considered not to be adverse.
During the lactation period mean food consumption was statistically significantly lower at this dose level when compared to the respective control value. Because no dose-dependent reduction of the mean food consumption through all dose levels was observed, the alteration observed at the mid dose was considered not to be test item-related but a result of biological variability.

Treatment of females with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption. From day 1 to 8 of the pre-pairing period mean food consumption at the high dose was 11.4 compared to 15.5 g/animal/day in the control group. After this period mean food consumption recovered and during the second week of the pre-pairing period was even higher than the respective control value (not statistically significant). Therefore the reduction of food consumption was considered not to be adverse. During the gestation period mean food consumption was similar to the respective control values.

The overall differences in mean food consumption of females at the dose levels of 100, 300 and 1000 mg/kg bw/day were respectively: +1.2%, -4.3% and -8.0% during the pre-pairing period, +1.4%, -3.7% and ±0.0% during the gestation period and -2.1%, -15.7% and +4.3% during the lactation period (percentages refer to the respective values in the control group).


1.4 BODY WEIGHTS OF MALES

See attached tables on pp. 12, 13, 19, 20, 26, 27

Pre-pairing and Pairing Periods

No effects on mean body weight gain and mean body weights were observed at the dose level of 100 mg/kg bw/day during the entire treatment period.

Treatment of males with the test item at the dose level of 300 mg/kg bw/day caused reduction of body weight gain during the pre-pairing period. This reduction was more pronounced at the beginning of the treatment (statistically significant from day 2 to 5 of the pre-pairing period). Afterwards body weight gain recovered and during the pairing period was similar to the respective control values. Alteration of the body weight gain had no apparent impact on the body weights of males. During the entire pre-pairing and pairing periods mean body weights at this dose level were similar to the respective control values. Therefore the reduction of body weight gain was considered not to be adverse.

Treatment of males with the test item at the dose level of 1000 mg/kg bw/day caused statistically significant reduction of body weight gain during the entire pre-pairing period. This resulted in a statistically significantly lower mean body weights noted from day 3 to 7 of the pre-pairing period. Both, body weight gain and body weights recovered and during the pairing period were similar to the respective control values. Therefore the reduction of body weight gain and reduction of body weights were considered not to be adverse.

The overall mean body weight gains in the control group and group treated with the test item at the dose levels of 100, 300 and 1000 mg/kg were respectively: +9.0%, +8.8%, +7.2% and +4.4% during the pre-pairing and +9.6%, +9.6%, +9.5% and +9.74% during the pairing periods (percentages refer to the respective time intervals).


1.5 BODY WEIGHTS OF FEMALES

See attached tables on pp. 14-18; 21-25; 28-30

Pre-pairing, Gestation and Lactation Periods

No effects on mean body weight gain and mean body weights were observed at the dose levels of 100 and 300 mg/kg bw/day during the entire treatment period.

Treatment of females with the test item at the dose level of 1000 mg/kg bw/day caused reduction of body weight gain during the pre-pairing period. This reduction was more pronounced at the beginning of the treatment (statistically significant from day 3 to 6 of the pre-pairing period) and resulted in the statistically significant reduction of the mean body weights noted from day 3 to 8 of the pre-pairing period. Afterwards body weight gain and body weights recovered and toward the end of the pre-pairing period as well as during the entire gestation and lactation periods were similar to the respective control values. Therefore the reduction of body weight gain and reduction of body weights were considered not to be adverse.

The overall mean body weight gains in the control group and group treated with the test item at the dose levels of 100, 300 and 1000 mg/kg were respectively: +7.0%, +7.0%, +7.1% and +7.1% during the pre-pairing, +57.1%, +53.7%, +48.1% and +57.1% during the gestation and +3.9%, +4.8%, +3.7%, and +6.5% during the lactation periods (percentages refer to the respective time intervals).


2 REPRODUCTION AND BREEDING DATA

2.1 MATING PERFORMANCE AND FERTILITY

No effects of the treatment with the test item on mating performance or fertility were observed at any dose level. Mean precoital times were 2.4, 2.7, 2.8 and 2.5 days in order of ascending dose levels.

One female (no. 50, paired with male no. 10) in the control group and 2 females (no. 63, paired with male no. 23 and no. 67, paired with male no. 27) at the dose level of 300 mg/kg bw/day did not mate during the first pairing period. All these females mated after the change of the male on the first day of the second pairing period.

Except for 1 female (no. 43, mated with male no. 3) in the control group and 1 female (no. 51, mated with male no. 11) at the dose level of 100 mg/kg bw/day, all females were pregnant and gave birth to live pups.

Consequently, fertility index and conception rate were 90% in the control group and at the dose level of 100 mg/kg bw/day and 100% at the dose levels of 300 and 1000 mg/kg bw/day whereas gestation index was 100% in all groups.


2.2 DURATION OF GESTATION

No effect of the treatment with the test item on duration of gestation was observed at any dose level. In order of ascending dose levels, mean duration of gestation was 21.1, 21.4, 21.4 and 21.4 days.


2.3 CORPORA LUTEA COUNT

No effect of the treatment with the test item on the number of corpora lutea was observed at any dose level.

The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups: 15.6, 14.0, 13.6 and 14.9 in order of ascending dose levels.


2.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS

No effects of the treatment with the test item on the number of implantation sites or incidence of pre- and post- implantation loss were observed at any dose level.

No significant differences in the number of implantation were observed. Mean number of implantation sites per dam were: 12.4, 11.2, 9.7 and 11.2 in order of ascending dose level.

No marked post-implantation loss was observed in any group. The mean incidence of post-implantation loss as a percentage of total implantations was 1.8%, 2.0%, 1.0% and 0.0% in order of ascending dose levels.


2.5 LITTER SIZE AT FIRST LITTER CHECK

No effect of the treatment on the number of live pups at first litter check was observed at any dose level.

The mean number of live pups per litter was similar in all groups: 12.2, 11.0, 9.6 and 11.2 in order of ascending dose level.


2.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

No effect of the treatment on the postnatal loss was observed at any dose level.

The total numbers of pup lost during the first 4 days of lactation were 3, 1, 12 and 3 in order of ascending dose levels, which corresponded to 2.7%, 1.0%, 12.5% and 2.7% of living pups, respectively.

The postnatal loss at the dose level of 300 mg/kg bw/day was statistically significantly higher when compared to the control group. However, all 12 pups lost at this dose level came from a single litter (no. 66). These pups were killed for ethical reason after termination of the parent female on day 3 post partum. Therefore the higher incidence of postnatal loss at this dose level was not test item-related.


3 TERMINAL FINDINGS - PARENTAL ANIMALS

3.1 ORGAN WEIGHTS

No effect of the treatment on the weights of testes and epididymides was observed at any dose level.

Mean values of the organ weights (absolute and relative to body weight) were similar in all groups.


3.2 MACROSCOPICAL FINDINGS

No test item-related macroscopical findings were noted at any dose level neither in males nor in females.

The incidences of testes reduced in size in 1 male (no. 3) and seminal vesicles reduced in size in 1 male (no. 9), both noted in the control group, were considered to be of spontaneous occurrence.


3.3 HISTOPATHOLOGY FINDINGS

A thorough examination of the reproductive organs both in males and females did not reveal any change related to the treatment with the test item.

No microscopical findings were noted in any female in the control group or at the high dose level as well as in the infertile female (no. 51) at the dose level of 100 mg/kg bw/day.

In males microscopical findings were found in 5 males in the control group, and 1 male each at the dose levels of 100, 300 and 1000 mg/kg bw/day. All these findings (listed below) were considered to be incidental.

In the control group:
- the infertile male no. 3 (with macroscopical changes) had pronounced atrophy and edema in the testes, aspermia and mixed cell infiltration in epididymides and a slight atrophy of prostate gland and seminal vesicles,
- male no. 2 had mixed cell infiltration in prostate gland,
- male no. 6 mixed cell infiltration in epididymides,
- male no. 9 (with macroscopical changes) had mixed cell infiltration in epididymides, slight atrophy of seminal vesicles and inflammation of prostate gland,
- the infertile male no. 10 had mixed cell infiltration in prostate gland and epididymides,

At the dose level of 100 mg/kg bw/day:
- the infertile male no. 11 had mixed cell infiltration in epididymides and prostate gland.

At the dose level of 300 mg/kg bw/day:
- the infertile male no. 23, had inflammation of prostate gland.
No findings were noted during the microscopical examination of the second infertile male (no. 27) at this dose level.

At the dose level of 1000 mg/kg bw/day:
- two males, nos. 32 and 34 had mixed cell infiltration in epididymides.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on observations the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for reproduction and development was considered to be 1000 mg/kg bw/day.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the dose level of 1000 mg/kg bw/day, body weight gain and body weights of pups were lower then the respective control values. Because of the minor nature of this effect it was considered not to be adverse.

CONCLUSION: No adverse effects

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

1 LITTER DATA - F1 PUPS

1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

No findings were noted at first litter check at any dose level.


1.2 SEX RATIOS

No effect of the treatment on the fetal sex ratio were noted at any dose level.

The proportion of male pups at first litter check was 54%, 42%, 51% and 47% in order of ascending dose levels.


1.3 PUP WEIGHTS TO DAY 4 POST PARTUM

See attached tables on pp. 31, 32

No effect of the treatment on pup body weights on days 1 and 4 post partum were noted at the dose levels of 100 and 300 mg/kg bw/day. Also body weight gain of pups during the 4 days of the lactation period at these dose levels was similar to the control values.

At the dose level of 1000 mg/kg bw/day, body weights of pups on day 1 post partum were by 3.4% lower then the respective control value. Because the body weight gain of pups during the first 4 days of the lactation period was lower at this dose level, the difference between the body weights of pups at the high-dose and in the control group increased. Consequently, mean body weights of pups on day 4 post partum at the high-dose was by 6.0% lower than the respective control value. The effects on body weight gain and body weights of pups at this dose level were not statistically significant. The possibility that they are caused by the treatment of the parent females with the test item was not excluded. Because of the minor nature of this effect it was considered not to be adverse.

On day 1 post partum mean body weights of pups were 5.8, 6.0, 6.0 and 5.6 g in order of ascending dose levels.
Differences in mean pup body weight gain from day 1 to 4 of the lactation period were +44.8%, +46.7%, +46.7% and +41.1% in order of ascending dose levels.


1.4 MACROSCOPICAL FINDINGS

No macroscopical findings were noted during the necropsy of pups at any dose level.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated (A)	10	10	10	10
Number of pregnant females (B)	9	9	10	10
Number of females which reared their pups until day 4 post partum (C)	9	9	9	10

(A)  Female nos. 50, 63 and 67 were mated during the second pairing period

(B)  Female nos. 43 and 51 were not pregnant.

(C)  Female no. 66 and its pups were killed for ethical reason on day 3 post partum. 

See attached document for tables of results.

Conclusions:

Based on the results of this study, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for the reproduction and development was considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the substance on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 10 males and 10 females were treated by gavage with the substance once daily. Males were treated over a 14-day pre-pairing period and during the pairing periods up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

Group 1:                0 mg/kg body weight/day (control group)

Group 2             100 mg/kg body weight/day

Group 3:            300 mg/kg body weight/day

Group 4           1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 300).

 

The following results were obtained:

 

 

PARENTAL ANIMALS

 

Mortality and General Tolerability

 

No test item-related deaths occurred during the study.

 

No clinical signs were observed in the control group and at the dose level of 100 mg/kg bw/day.

 

At the dose levels of 300 and 1000 mg/kg bw/day in both sexes pushing head through the bedding material and salivation after application were observed during the most of the treatment period. These symptoms were considered to be signs of discomfort caused by the treatment with the test item but not adverse effect. A short-lasting diarrhea was observed in males at the dose levels of 300 and 1000 mg/kg bw/day and females at the dose level of 1000 mg/kg bw/day and activity reduced for 1 to 3 days was observed in males at the dose level of 1000 mg/kg bw/day.

These symptoms were considered to be test item-related but not adverse effects.

 

Food Consumption

 

Treatment with the test item at the dose levels of 100 mg/kg bw/day caused no effects on food consumption neither in males nor in females.

 

Treatment with the test item at the dose level of 300 mg/kg bw/day caused in females a not statistically significant reduction of mean food consumption at the beginning of the treatment period. Afterwards mean food consumption increased and was similar to the respective control values.

 

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption in males and females at the beginning of the treatment. Afterwards mean food consumption increased and was similar (in males) or even slightly higher (in females) than the respective control values.

 

Because food consumption recovered after the initial reduction and in all groups remained similar to the respective control values until the completion of the treatment, the reduction of food consumption was considered not to be adverse.

 

Body Weights

 

Treatment with the test item at the dose level of 100 mg/kg bw/day caused no effects on body weight gain and body weights neither in males nor in females.

 

Treatment with the test item at the dose level of 300 mg/kg bw/day caused in malesa statistically significant reduction of body weight gain at the beginning of the treatment period. Afterwards body weight gain increased and was similar to the respective control values.

 

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of body weight gain in males and females at the beginning of the treatment. This resulted in a statistically significant reduction of body weights in both sexes. Afterwards body weight gain and body weights increased in both sexes and were similar to the control values during the remaining treatment period.

 

Because body weight gain and body weights recovered after the initial reduction and in all groups remained similar to the respective control values until the completion of the treatment, the reduction of body weight gain and body weights was considered not to be adverse.

 

Reproductive Data

 

The relevant reproductive data: mating performance, fertility, gestation, number of corpora lutea, implantation rate and post-implantation loss were not affected by the treatment with the test item.

 

Organ Weights

 

No effects on the weight of testes or epididymides were observed at any dose level.

 

Macroscopical Findings and Histopathological Examinations

 

No test item-related macroscopical findings were noted at any dose level neither in males nor in females.

 

LITTER DATA - F1 PUPS

 

No clinical signs were noted at first litter check and during the 4 days of the lactation period.

 

No macroscopical findings were noted during the necropsy of pups at any dose level

 

No effects on body weights of pups were observed at dose levels of 100 and 300 mg /kg bw/day.

 

At the dose level of 1000 mg/kg bw/day, body weight gain and body weights of pups were lower then the respective control values. The effects were not statistically significant. The possibility that the treatment of the parental females with the test item at this dose level caused a disturbance in the body weight of their pups was not excluded. Because of the minor nature of this effect it was considered not to be adverse.

Based on the results of this study, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for the reproduction and development was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Reproduction/Developmental screening test performed according to the OECD Guideline 421 and in compliance with GLP, four groups of 10 males and 10 females were treated by gavage with AA 15 once daily at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were treated over a 14 -day pre-pairing period and during the pairing periods up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

Parental results:

No test item-related deaths occurred during the study.

No clinical signs were observed in the control group and at the dose level of 100 mg/kg bw/day.

At the dose levels of 300 and 1000 mg/kg bw/day in both sexes pushing head through the bedding material and salivation after application were observed during the most of the treatment period. These symptoms were considered to be signs of discomfort caused by the treatment with the test item but not adverse effect. A short-lasting diarrhea was observed in males at the dose levels of 300 and 1000 mg/kg bw/day and females at the dose level of 1000 mg/kg bw/day and activity reduced for 1 to 3 days was observed in males at the dose level of 1000 mg/kg bw/day.

These symptoms were considered to be test item-related but not adverse effects.

Food consumption and body weights were reduced at 300 and 1000 mg/kg bw/day with a statistically difference at 1000 mg/kg bw/day. These reductions were considered not to be adverse.

Mating performance, fertility, gestation, number of corpora lutea, implantation rate and post-implantation loss were not affected by the treatment with the test item.

No effects on the weight of testes or epididymides and no macroscopical or histopathological findings were noted at any dose level.

Litter results:

No clinical signs and macroscopic findings were observed in pups, at any dose level.

No statistically significant effects on body weights and body weight gain were noted at any dose level.

NOAEL for general parental toxicity and for the reproduction and development was considered to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Prenatal Developmental Toxicity Dhandapani P 2023.

This study was performed to investigate the effects of prenatal exposure on pregnant test animals and on the developing organism by the repeated oral exposure of the test item AMIDET A 15 from impregnation to one day prior to expected parturition.

Ninety-four mating-confirmed female Wistar rats were assigned to four groups viz., Vehicle control (G1), Low dose (G2), Intermediate Dose (G3) and High dose (G4). Groups G1 and G2 consisted of 24 female rats,  Groups G3 and G4 consisted of 23 female rats on GD0. 22 females were found to be pregnant in G1 and G2 group out of 24, 23 in G3 group and 22 were pregnant in G4 group on GD20. The test item, Amidet A 15, was formulated as a suspension using polyethylene glycol 300 as the vehicle. Dose formulations were administered at dose levels of 100 (G2), 300 (G3), and 1000 (G4) mg/kg/day and the dose volume was maintained at 5 mL/kg body weight. The vehicle polyethylene glycol 300 was administered to the vehicle control (G1) group at a volume of 5 mL/kg body weight.

The identity of the test item was based on the Certificate of Analysis (CoA) provided by the Sponsor. The test item formulations were found to be stable for 7 days when stored at room temperature and homogeneous in polyethylene glycol 300. The results of dose formulation analyses carried out before initiation of treatment and towards the end of treatment period showed that the mean concentration of the test item was within acceptable limits (90-110% of recovery for the nominal concentration).

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination. Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Test item-related clinical findings like salivation and pressing and pushing the head through the bedding material were noted in 4 females in 300 mg/kg b.w. and all females in the 1000 mg/kg b.w. groups at the daily examinations post dosing. All these animals recovered from the clinical signs of toxicity after approximately 10-15 mins following dosing. All the animals of G1 and G2 dose groups were observed to be normal throughout the experimental period. No bleeding or abortion was observed on GD5 following dosing.

No test item-related findings were noted in fetus weight, ano-genital distance (AGD), AGD index and crown crump length. A statistically significant increase in crown crump length in G2 group female fetuses was observed. A statistically significant increase in fetus weight and decrease in AGD index were observed in G3 group male fetuses. A statistically significant increase in fetal weight, and decrease in crown crump length and increase in AGD index was observed in G3 group female fetuses. These changes were all considered as non-dose dependant and sporadic. Intrauterine growth and survival in the 100, 300, and 1000 mg/kg b.w. groups were unaffected by test item exposure. One fetus in the 300 mg/kg b.w group and one fetus in the 1000 mg/kg b.w had absence of tail, which were considered to be unrelated to exposure because the incidence was within the published historical range for the rat.

Body weights of the dams during the gestation period and the gestational body weight change corrected for gravid uterine weight was comparable between the control and treatment groups. No test item related changes were observed in feed consumption of treatment group animals when compared with the control group animals. No gross pathological changes were observed in any of the dams of control or treatment groups. 

Uterine content examination of the dams revealed no abnormalities. The gravid uterine weight, number of corpora lutea and implantation sites, and pre-and post-implantation loses were similar between control and treatment groups. The number of early resorptions and live fetuses and the percent viable fetus per litter were comparable between the treatment and the control groups.

No soft tissue alterations were evident in the visceral examination of the fetuses. No test item related major or minor skeletal malformations were observed in the skeletal examination of fetuses. The normal skeletal variations that were observed in the fetuses of all groups were of no toxicological significance.

No test item related changes were observed in the thyroid weights and in the histopathological examination of thyroids and no changes in thyroid hormone levels were observed in the treatment groups in comparison to the control group.

In conclusion:

Based on the results obtained in the current study, it is evident that pregnant Wistar rats, exposed to the test item from impregnation (gestation day 0) until one day prior to their expected day of parturition (gestational day 20) at the dose level of 100, 300, and 1000 mg/kg b.w. did not show any evidence for maternal or developmental toxicity. Hence it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats. 

Furthermore, in a Pre-natal Developmental Toxicity Study (read-across, OECD 414, GLP, Key, rel.1): NOAEL(maternal and developmental) = 1000 mg/kg bw/day in rats. No systemic adverse effect reported in the dams and no effect in foetuses at the highest dose level tested in this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2021 to 03 February 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

OECD Guideline for Testing of Chemicals: Prenatal Developmental Toxicity Study, No. 414, Adopted: 25th June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of the Test Item: AMIDET A 15

Description: White to off-white powder


Batch No.: 30905893

Date of Production: 01.02.2021

Date of Expiry: 24.01.2022

Date of Receipt: 08.05.2021

Sponsored by: Kao Chemical Europe, S.L.
Puig dels Tudons 10 –
08210 BARBERÀ DEL VALLÈS (Barcelona)
SPAIN

Species:

rat

Strain:

Wistar

Remarks:

Species: Rat (Rattus norvegicus) Strain : Wistar (Crl:WI)

Details on test animals or test system and environmental conditions:

No. of animals
(Range finding study) : 28 animals (14 males +14 females)


No. of animals (Main study): 150 animals (50 males and 100 females)

Source: Hylasco Bio-Technology (India) Pvt. Ltd.
No. 4B MN Park, Turkapally Village,
Shameerpet Mandal, Medchal District,
Hyderabad, Telangana 500 078.
Location of the Study

The study was conducted in the experiment unit-I, room no. 4 at the animal house facility, IIBAT, Padappai - 601 301, Tamil Nadu, India.

Animal House Condition
Temperature was maintained between 21.1 and 22.8 °C, and the relative humidity between 48 and 60%. Temperature and relative humidity were recorded once daily. The test room was provided with 12-hour artificial light and 12-hour dark condition.

Housing
Animals were housed in clean, sterilized solid floored standard polypropylene rat cages covered with a stainless steel grill with provisions for feed and water. Cages were placed on stainless steel racks. Cage rotation was done at weekly intervals. Clean and sterilized corn cob was used as bedding material. During the acclimatization period, animals were housed in groups of two/three per cage. After the acclimatization period, one female was housed with one male until the confirmation of mating (for a maximum period of 14 days) in cages equipped with stainless steel bottom grills with blotting paper underneath. After the confirmation of mating, the females were housed individually. Clean and sterile nesting materials (Nestlets) was provided to all dams from gestation day (GD) 14 until scheduled sacrifice on GD 20. Male animals housed individually during the mating period, and females housed individually during the gestation period were provided with the enrichment devices (tunnels).

Sanitation
The floor of the experimental rooms was swept and mopped daily. Cages, enrichment devices, nesting materials and bedding material were changed twice weekly and water bottles were changed daily. All the experimental procedures were done in a clean environment.

 
Animal Welfare and Approval

IIBAT is committed to enhance animal welfare and registered with CPCSEA, Ministry of Environment and Forests, Government of India for Breeding and experiments of Animals (No.31/PO/RcBi - S/RcBi - L/1999/CPCSEA dated 11 May 2017). Use of live animals is inevitable to accomplish the purpose of this study. The recommendations regarding animal care and handling were followed and consistent with:

Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for Laboratory Animal Facility, The Gazette of India, 2018.

The use of animals and the general procedures involved in the present study have been reviewed and approved by the Institutional Animal Ethics Committee of IIBAT (Approval Number: 25/155/IAEC/IIBAT/2021; dated 29.05.2021).

Feed and Water
Standard and conventional laboratory rodent pellet feed (manufactured by Special Diet Services - England, supplied by Vivo Biotech Ltd., Hyderabad, India), and potable well water (IIBAT) processed through reverse osmosis was provided ad libitum to all animals. Phytooestrogen levels in the feed and bedding material was tested and was ensured to be within the acceptable limits (6.94 mg/kg of Genistein and 0.50 mg/kg in the rodent laboratory feed and bedding material respectively). Analysis reports on phytoestrogen levels in feed and bedding material, microbial load and chemical contaminants in feed and water, and nutrient content of feed are retained with the facility records and an authorized photocopy was maintained with the raw data.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol (PEG) 300 was selected as vehicle for test item preparation. It was selected based on the available information on the test item (ECHA, 2010)). A solubility check was performed using the test item prior to initiation of dosing period.

Details on exposure:

Preparation of Test Item Formulation and Administration of Doses

Dose formulations were prepared by mixing the test item with Polyethylene glycol (PEG) 300 at the desired concentrations. The required test item formulations were prepared approximately 30 minutes prior to the start of dosing of animals. The test item was administered at approximately the same time (±1 h) each day as a single dose orally by gavage using a stainless steel oral intubation needle fitted to a graduated syringe. The dose volume was maintained at 0.5 ml/100 g body weight (b.w.). Animals in all the groups were administered the same volume of either the test item formulation or vehicle. Vehicle control group received the vehicle in the highest amount used as in the low dose group.

Dose Formulation Analysis

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21140) at the Analytical Chemistry Department of IIBAT.



Study Design

Range finding study
Attached as appendix - 1

Main Study

The main study was conducted using three treatment groups and a vehicle control group. Mating procedure was followed as described in section Mating of animals, 50 males and 100 females targeted to yield a minimum of 20 pregnant rats to maximum 25 pregnant rats per group at the end of mating period. Pregnancy-confirmed females were randomly assigned to the groups to obtain the best possible distribution of day 0 gestation weights between groups and were dosed from GD 0 until GD 19. The dose levels for the main study were determined based upon the results of the range finding study. The animals were dosed from GD 0 till GD 19 based on the most recent individual body weights. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations. Males used for mating were not dosed in the study and were sacrificed at the end of the mating period on day 15.

The high dose was selected with the aim to induce some developmental and/or maternal toxicity but not death or severe suffering, an intermediate dose with minimal observable toxic effects, and a low dose with no evidence of either maternal or developmental toxicity. The dose level used in Group 4 was the maximum dose level recommended in the OECD 414 test guideline. Animals in the control group was handled in an identical manner to the test group animals, except that they were administered with vehicle alone.


Groups Dose levels Number of pregnant females per group*
G1 (Vehicle Control) 0 mg/kg b.w. 24
G2 (Low dose) 100 mg/kg b.w. 24
G3 (Intermediate dose) 300 mg/kg b.w. 23
G4 (High dose) 1000 mg/kg b.w. 23
*Based on the pregnancy status of the animals ascertained during terminal sacrifice on GD 20 (Refer appendix – 1 & 2).

OBSERVATIONS

Animals were observed at least once daily throughout the study period. All observations related to this study were recorded manually in the respective formats for raw data collection.

Morbidity/Mortality

Animals were observed for morbidity/mortality once daily from the day of acclimatization until the experiment completion.

Clinical Observations

General cage-side clinical observations were performed once daily in the animals. Pertinent behavioral changes, and all signs of toxicity, including mortality, morbidity were recorded in terms of time of onset, degree and duration of toxicity signs if any were also recorded. During pregnancy, maternal animals were monitored for signs of abortion or premature delivery, if any.

Body Weight

During gestation, in the main study females were weighed on GD 0, 5, 8, 11, 14, 17, and on GD 20 prior to terminal sacrifice.

Feed Consumption

The feed consumption of the animals during the main study was calculated for gestation days 0 to 5, 6 to 8, 9 to 11, 12 to 14, 15 to 17, and 18 to 20. The feed consumption was calculated from the feed input and leftover feed data and reported as g/rat/day. The feed consumption was also recorded during the mating period.

The cage-wise feed consumption was estimated by following formula:

Food consumption (g/rat/day)=(Food consumption during interval per cage (g))/(No.of days in the interval)

Blood sample collection and analysis of thyroid hormones

Blood samples was collected individually from all dams on gestation day 20 and all non-pregnant females individually at scheduled termination day (GD 20). The blood samples were drawn from the animals under isoflurane anesthesia, from the retro - orbital plexus without anticoagulant. The serum was separated from the blood and stored at -20 °C until analysis. Blood samples were collected at approximately the same time (± 30 min) at each day.

Blood samples were analyzed for thyroid hormones triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) using an ELISA method.

Pathology

Gross pathology examination

In the study, all surviving dams were humanely euthanized on GD 20 by carbon dioxide asphyxiation and were subjected to detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Evaluation of the dams during caesarean section and subsequent fetal analyses and sample collection were done randomly across available groups to avoid sampling bias.

Examination of Uterine Contents

During termination or as soon as possible after death, the uteri of the animal were removed and the pregnancy status of the animals were ascertained. Uteri that appear non-gravid were examined further by ammonium sulphide staining to confirm the non-pregnant status. Gravid uteri including the cervix were weighed. Gravid uterine weights were not recorded from animals found dead during the study. The number of corpora lutea were determined in pregnant animals. The uterine contents were examined for number of embryonic or fetal deaths and viable fetuses. The degree of resorption was described (early, late) to estimate the relative time of death of the conceptus. The number of implantation sites were counted in females .

Based on the results, the following was calculated (Hong, 2014):


Preimplantation loss (%)= ((number of corpora lutea - number of implantations))/((number of corpora lutea) )×100

Postimplantation loss (%)= ((number of implantations - number of live fetuses))/((number of implantations) )×100


Fetal death =resorptions + dead fetuses

Examination of Fetuses

During termination live fetuses was euthanized by placing on a pre-chilled ice-cold plate covered with paper towel and marked with an appropriate identification number. Fetuses were examined externally for gross abnormalities. Particular attention was paid to the external genitals which were examined for signs of altered development of the fetuses. Each live fetus was measured for its crown-rump length, weighed, sexed and marked individually soon after termination of dam. Each fetus was examined at termination to establish the number and sex of fetus, stillbirths, live births, runts (fetuses that are significantly smaller than corresponding control fetus) and the presence of gross anomalies and/or other abnormalities.

The anogenital distance (AGD) of each live fetus was measured using a calibrated digital Vernier caliper with 0.01 mm accuracy. For male fetus AGD was measured from the anterior edge of the anus to the base of the anogenital aperture, and for female fetus, it was measured from the anterior edge of the anus to the base of the urinary aperture. AGD were normalized to a measure of fetus size (cube root of body weight) by calculating the AGD Index.

AGD Index = AGD of live fetus/Cube root of body weight
Fetuses were examined for skeletal and soft tissue alterations including variations and malformations or anomalies.

External fetal sex determined by gross examination was compared with internal (gonadal) sex in all fetuses examined for both skeletal and soft tissue malformations.

Approximately one-half of each litter was prepared for skeletal alterations. The remaining half of the litter was prepared and examined for soft tissue alterations. The alternate fetuses were selected for skeletal examination starting with the first fetus (closest to the right ovary) in even-numbered dams and the second fetus in the odd-numbered dams (Narotsky, 2003).

Visceral (Internal)

Fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. Fetal kidneys were examined and graded for renal papillae development. This examination includes the heart and major vessels. The sex of all fetuses was confirmed by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter were examined by a mid-coronal slice.

Skeletal Examinations

All carcasses, including the carcasses without heads, was eviscerated and fixed in alcohol and processed for staining of the skeletal and cartilaginous tissues using Alizarin Red S and Alcian Blue. These specimens were cleared in potassium hydroxide solution and, following staining, they were stored in glycerin for subsequent examination of the skeletons.

Organ weighing and Histopathological examination

Organ weighing was performed post fixation for thyroid gland in dams. Thyroid glands of all dams were preserved in 10% neutral buffered formalin for histopathological examination.

Detailed histological examination of the thyroid gland was performed on the dams of all treatment groups and the control group.

Disposal of waste generated during the study

Waste generated during the study was disposed as per SOP/GEN/003.

STATISTICAL ANALYSIS

Data from non-gravid females was excluded from calculation of means and from comparative statistics.
Continuous data variables such as maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone assessment data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative to body weight), anogenital distance, and crown-to-rump length was initially analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity was detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test were significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify the group which shows a significant difference from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss and the fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA test to determine the intergroup difference. If the results of the non-parametric ANOVA were significant (p<0.05), the Mann-Whitney-U test was performed, where appropriate, to identify the group which shows a significant difference from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and developmental variations was subjected to the Kruskal-Wallis non-parametric ANOVA test followed by Mann-Whitney U test (if appropriate) as described above.

A p-value of <0.05 was considered statistically significant. The analysis was performed using IBM SPSS Statistics, version 28.

Analytical verification of doses or concentrations:

yes

Remarks:

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period.

Details on analytical verification of doses or concentrations:

Dose Formulation Analysis

The dose formulation was subjected to homogeneity, stability and active ingredient analysis once prior to the initiation of dosing and once during the last week of the dosing period. The sampling was performed from the top, middle, and bottom of the container with dose formulations in duplicate preparations for three dose levels. The analysis was performed by validated analytical method (IIBAT study no. 21140) at the Analytical Chemistry Department of IIBAT.

Details on mating procedure:

Mating of animals

Mating of animals was done in two batches. After 5 days of acclimatization, for the first batch of mating, 50 male and 50 female animals were randomly cohabited in 1:1 ratio (male:female). After confirming mating, male animals were separated and returned to their respective cage and the second batch of mating with 50 female animals was continued as mentioned above. The males were humanely euthanized by carbon dioxide asphyxiation after the last day of mating period.

Once daily until evidence of copulation, the female animals were examined for presence of vaginal plug, or sperm by vaginal smear examination. Day 0 (GD 0) of pregnancy is defined as the day on which mating evidence (vaginal plug, or sperm in the vaginal smear) was found. The mating of animals was continued for 7 days, if vaginal plug or sperm was not observed. If the mating was not successful even after 7 days, the females were remated with proven males / fresh males. The remated pairs were observed for another 7 days and were confirmed for gestation by following the above procedure. The females mated by same male were distributed among the groups. Females showing no-evidence of copulation were humanely euthanized by carbon dioxide asphyxiation after the last day of mating period (after a maximum of 14 days of mating period). Female Mating Index (measure of female’s ability to mate) and Female Fertility Index (measure of female’s ability to become pregnant) was calculated as follows:

Mating index of untreated females (%)= ((No.of untreated females mated))⁄((no.of untreated females placed with untreated males)) × 100
Female fertility index (%) = (No. of females pregnant/no. of females with confirmed mating) × 100.

Randomization
Females with confirmed mating (day 0 of gestation, GD 0) were randomly assigned to the groups using a stratified body weight distribution method (SOP/TOX/001) to equalize as best possible the gestation day 0 weights between groups, and were identified using ear-tags bearing a unique animal number. The body weight range among the animals used at each dose level did not exceed ± 20 % of the mean weight before start of the treatment.

Duration of treatment / exposure:

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination. Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Frequency of treatment:

The test item, Amidet A 15, was formulated as a suspension using polyethylene glycol 300 as the vehicle. Dose formulations were administered at dose levels of 100 (G2), 300 (G3), and 1000 (G4) mg/kg/day and the dose volume was maintained at 5 mL/kg body weight. The vehicle polyethylene glycol 300 was administered to the vehicle control (G1) group at a volume of 5 mL/kg body weight.

Duration of test:

This study was performed to investigate the effects of prenatal exposure on pregnant test animals and on the developing organism by the repeated oral exposure of the test item AMIDET A 15 from implantation to one day prior to expected parturition.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (G1)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose (G2)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate Dose (G3)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose (G4)

No. of animals per sex per dose:

Ninety-four mating-confirmed female Wistar rats were assigned to four groups viz., Vehicle control (G1), Low dose (G2), Intermediate Dose (G3) and High dose (G4). Groups G1 and G2 consisted of 24 female rats, Groups andG3 and G4 consisted of 23 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Route of Exposure

The oral route was chosen as it is recommended route of exposure in the OECD test guideline 414, and it is one of the possible routes of exposure to humans.
Healthy young adult animals were selected for the study after a detailed clinical examination by a veterinarian. Only animals free of obvious health abnormalities were used for the study. Female animals were nulliparous and non-pregnant at the time of receipt for the study. The body weight range among the animals used for the experiment did not exceed ± 20 % of the mean weight of each sex at the start of the acclimatization.

Identification of Animals
In each cage, animals were identified with the numbers marked on their tail (during mating period) or by ear-tag bearing unique animal number (after confirmation of mating). The cages were identified by attaching a cage card containing information such as study number, cage number, animal number(s), species, sex, dose level, test item name, date of confirmation of mating along with the signature of study director or study personnel.

Acclimatization
All animals were acclimatized for a period of five days. Animals were weighed on the day of receipt and observed daily. Detailed records of acclimatization were maintained in the raw data.

Justification for the Choice of the Test System
The rat was selected because it has been historically proven to be a suitable test system for developmental toxicity studies, and rat is one of the preferred test systems as per the OECD test guideline 414.

Maternal examinations:

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery

Ovaries and uterine content:

Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination.

Blood sampling:

Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Fetal examinations:

visceral and skeletal examinations of fetuses, fetus weight, ano-genital distance (AGD), AGD index and crown crump length.

Statistics:

STATISTICAL ANALYSIS

Data from non-gravid females was excluded from calculation of means and from comparative statistics.
Continuous data variables such as maternal body weights, gravid uterine weights, weight changes, feed consumption data, thyroid hormone assessment data, fetal body weight (separately by sex, and combined), organ weights (absolute and relative to body weight), anogenital distance, and crown-to-rump length was initially analyzed for normality using Shapiro-Wilk test, and homogeneity of variance between groups was checked by Levene’s test. Where significant homogeneity was detected, a one-way analysis of variance (ANOVA) was carried out followed by parametric multiple comparison procedures using Dunnett's test for post hoc comparison. For non-normally distributed data, non-parametric ANOVA, Kruskal-Wallis test was used. If the results of the test were significant (p<0.05), the Mann-Whitney U test or Dunn’s test (pairwise comparison) was performed to identify the group which shows a significant difference from control.
The group mean numbers of corpora lutea, implantation sites, viable fetuses, the mean litter proportions of prenatal data (% per litter of viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss and the fetal sex distribution) was analysed using Kruskal-Wallis, non-parametric ANOVA test to determine the intergroup difference. If the results of the non-parametric ANOVA were significant (p<0.05), the Mann-Whitney-U test was performed, where appropriate, to identify the group which shows a significant difference from control.

The mean litter proportion (% per litter) of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation was tabulated. The mean litter proportions of fetal malformations and developmental variations was subjected to the Kruskal-Wallis non-para

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical findings like salivation and pressing and pushing the head through the bedding material were noted in 4 females in 300 mg/kg b.w. and all females in the 1000 mg/kg b.w. groups at the daily examinations post dosing. All these animals recovered from the clinical signs of toxicity after approximately 10-15 mins following dosing. All the animals of G1 and G2 dose groups were observed to be normal throughout the experimental period. No bleeding or abortion was observed on GD5 following dosing.

No premature deliveries or signs of abortion were observed in any of the dams of the treatment or control groups. All the remaining animals in the study were found to be normal throughout the experiment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two female animals exhibited mortality prior to scheduled termination, one in G1 and one in G2. No Mortality/morbidity was observed in any of the remaining animals pertaining to the treatment or control group throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of the dams measured at various time points during the gestation period (GD 0, 5, 8, 11, 14, 17 and 20) were comparable between the control and treatment groups. Body weight change during gestation (weight of the dams on GD20 minus the weight of the dams during GD0) and the gestational body weight change corrected for gravid uterine weight (body weight change during gestation minus the gravid uterine weight) were also comparable between the control and treatment groups (Table - 1).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption measured at various intervals during the gestation period (GDs 0-5, 6-8, 9-11, 12-14, 15-17 and 18-20) was comparable between the control and treatment groups and no statistical significance was observed (Table-2).

Endocrine findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weight (Thyroid)

The absolute and relative thyroid organ weights were comparable between the control and treatment groups (Table – 6).

Gross pathological findings:

no effects observed

Description (incidence and severity):

All the surviving dams were euthanized on GD 20 for gross pathological observation. None of the dams showed any signs of abortion or premature delivery prior to the scheduled termination on GD 20, and there was no premature delivery in the dams. Detailed examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents had revealed no abnormalities. No gross lesions were observed in the uterus and endometrial area between each implant site. No premature delivery was observed in the dams.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item related histopathological findings were observed in the thyroid organs of the treatment groups of animals. The histopathological findings observed in the study, such as colloidal alteration and degeneration with mononuclear infiltration in the thyroid follicles, were considered to be spontaneous or incidental and not related to the test item treatment (Brändli-Baiocco et al., 2018) (Table – 7).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Examination of Uterine Contents

At the termination (GD20), the uteri of the animals were removed, and the pregnancy status of the animals was ascertained. Uteri that appear non-gravid were stained with 10% (v/v) ammonium sulphide solution to confirm the non pregnant status of the animals. Out of 94 animals which showed the evidence of copulation, 85 were found to be pregnant, and 9 were non-pregnant. No stained implantation sites were observed in the uteri of non-pregnant animals. No variations were observed in the colour or volume of the amniotic fluid in the uterus of any dams.

Gravid uterine weight

The mean gravid uterine weight of treatment and control groups were similar. No test item-related findings were noted in corpus luteum count, implantation sites, total resorptions and total live fetus (Table – 4 and 8).

Thyroid hormones

Blood samples were analyzed for thyroid hormones (T3, T4, TSH) on GD 20 and the results were comparable between control and treatment groups. No test item-related findings were noted in absolute and relative thyroid organ weights.
No test item-related findings were noted in T3 and T4 hormones. A slight increase in TSH in G3 group was noted. This change was considered as non-dose dependent and incidental. During the experimental phase clinical signs such as lethargy, salivation and pressing the head inside the bedding material (due to irritation caused by the test item) was observed in the high dose group. (Table – 3 and 7).

Details on results:

Mating of animals

Out of 100 females were cohabitated with males in 1:1 ratio (male: female), evidence of copulation (confirmation of mating) was observed in 94 females. Female mating Index (measure of female’s ability to mate) was calculated as 94.00%. Mating was confirmed by the presence of vaginal plug in 94 females. Out of 94 females with confirmed evidence of mating, 94 showed the evidence of copulation within 7 days. 6 females which did not show evidence of copulation were humanely sacrificed by carbon dioxide asphyxiation after a maximum of 14 days of mating period.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea, implantations sites, and mean percent pre-and post-implantation losses were similar between control and treatment groups (Table – 4).

Early or late resorptions:

no effects observed

Description (incidence and severity):

In all dams the aminotic fluid was normal in colour, no gross lesions were observed in the endometrial areas between each implants. No late resorptions or dead fetuses were observed in any of the dams of treatment and control groups. The mean number of early resorptions, mean number of live fetuses and the percent viable fetus per litter were similar between treatment and control groups (Table – 4).

Dead fetuses:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Hence it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Remarks on result:

other: Hence it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups (Table-5 and 6).

Reduction in number of live offspring:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups (Table-5 and 6).

External malformations:

no effects observed

Description (incidence and severity):

No remarkable soft tissue alterations were observed in the any of the fetus of the treated dams or control dams (Table – 5).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In terms of skeletal examination, there was no incidence of major malformations observed in the fetuses of the treated group dams. Variants were observed in a minimal number of fetuses of the control and the treated dose group. Variations such as rudimentary ribs, bent ribs, short ribs and variation in the caudal centrum (0-5) were observed in the control and the treated groups. No anomalies were observed in the centrum of the vertebrae and the number of cervical, thoracic, lumbar and sacral vertebrae in the control and treated group fetuses. These normal variants were spontaneous and considered to be not due to the test item treatment, and have no toxicological significance. The incidence of malformations (major/minor) and normal variants in the fetuses were statistically similar between treatment and control groups. Caudal centrum ranging from 0 to 5 in the fetus of the control and the treatment groups is within normal limits (Table – 5).

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of the fetuses showed that Test item administered at the dose rate of (100 mg, 300 mg, 1000 mg/ kg body weight) did not cause any major abnormalities in any fetus of the treated dams. The external examinations pertaining to the length, cranium, eyes, limbs, tail, genitals and sex did not reveal any major abnormalities. The only finding observed was absence of tail in one fetus (2632-L4) in G3 group and one fetus (2646- L11) in G4 group . There was no external malformation observed in the any of the fetus of the high dose group. These findings are considered to be of spontaneous incidence and not related to treatment. All these findings were incidental and is of no toxicological significance (Table – 5).

Key result

Dose descriptor:

NOAEL

Remarks:

it is concluded that the no observed adverse effect level (NOAEL) for maternal and developmental effects for the test item AMIDET A 15 is greater than 1000 mg/kg b.w. to the Wistar rats.

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: External examination of the fetuses

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of the fetuses showed that Test item administered at the dose rate of (100 mg, 300 mg, 1000 mg/ kg body weight) did not cause any major abnormalities in any fetus of the treated dams. The external examinations pertaining to the length, cranium, eyes, limbs, tail, genitals and sex did not reveal any major abnormalities. The only finding observed was absence of tail in one fetus (2632-L4) in G3 group and one fetus (2646- L11) in G4 group . There was no external malformation observed in the any of the fetus of the high dose group. These findings are spontaneous incidence and not related to treatment. All these findings were incidental and is of no toxicological significance

The mean values of fetal weight (either separated by sex, or combined), anogenital distance, anogenital index, and crown-to-rump length were similar between treatment and control groups. The fetal sex was found to be evenly distributed among the control and treatment groups

No remarkable soft tissue alterations were observed in the any of the fetus of the treated dams or control dams

In terms of skeletal examination, there was no incidence of major malformations observed in the fetuses of the treated group dams

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Conclusions:

Based on the results obtained in the current study, it was evident that the exposure of the test item to pregnant Wistar rats, from impregnation (gestation day 0) until one day prior to their expected day of parturition (gestational day 20), at the dose levels of 100, 300, and 1000 mg/kg b.w. did not cause any maternal or developmental toxicity. Hence it was concluded that the no observed adverse effect level (NOAEL), for both maternal toxicity and developmental effects, for the test item AMIDET A 15 was greater than 1000 mg/kg b.w. in the Wistar rat.

Executive summary:

This study was performed to investigate the effects of prenatal exposure on pregnant test animals and on the developing organism by the repeated oral exposure of the test item AMIDET A 15 from impregnation to one day prior to expected parturition.

Ninety-four mating-confirmed female Wistar rats were assigned to four groups viz., Vehicle control (G1), Low dose (G2), Intermediate Dose (G3) and High dose (G4). Groups G1 and G2 consisted of 24 female rats,  Groups G3 and G4 consisted of 23 female rats on GD0. 22 females were found to be pregnant in G1 and G2 group out of 24, 23 in G3 group and 22 were pregnant in G4 group on GD20. The test item, Amidet A 15, was formulated as a suspension using polyethylene glycol 300 as the vehicle. Dose formulations were administered at dose levels of 100 (G2), 300 (G3), and 1000 (G4) mg/kg/day and the dose volume was maintained at 5 mL/kg body weight. The vehicle polyethylene glycol 300 was administered to the vehicle control (G1) group at a volume of 5 mL/kg body weight.

The identity of the test item was based on the Certificate of Analysis (CoA) provided by the Sponsor. The test item formulations were found to be stable for 7 days when stored at room temperature and homogeneous in polyethylene glycol 300. The results of dose formulation analyses carried out before initiation of treatment and towards the end of treatment period showed that the mean concentration of the test item was within acceptable limits (90-110% of recovery for the nominal concentration).

During the in-life phase of the study, from GD 0 till GD 19, all the dams were observed for morbidity/mortality, clinical signs of toxicity, body weight, feed consumption and for signs of premature delivery. Dams were sacrificed on GD 20 and were subjected for gross pathological evaluations, and examination of uterine contents, including the external, visceral and skeletal examinations of fetuses. Thyroid glands collected from the dams were weighed and subjected to histopathological examination. Blood samples collected from the dams during the terminal sacrifice (GD 20) were analyzed for thyroid hormones (T3, T4, TSH).

Test item-related clinical findings like salivation and pressing and pushing the head through the bedding material were noted in 4 females in 300 mg/kg b.w. and all females in the 1000 mg/kg b.w. groups at the daily examinations post dosing. All these animals recovered from the clinical signs of toxicity after approximately 10-15 mins following dosing. All the animals of G1 and G2 dose groups were observed to be normal throughout the experimental period. No bleeding or abortion was observed on GD5 following dosing.

No test item-related findings were noted in fetus weight, ano-genital distance (AGD), AGD index and crown crump length. A statistically significant increase in crown crump length in G2 group female fetuses was observed. Astatistically significant increase in fetus weight and decrease in AGD index were observed in G3 group male fetuses. A statistically significant increase in fetal weight, and decrease in crown crump length and increase in AGD index was observed in G3 group female fetuses. These changes were all considered as non-dose dependant and sporadic. Intrauterine growth and survival in the 100, 300, and 1000 mg/kg b.w. groups were unaffected by test item exposure. One fetus in the 300 mg/kg b.w group and one fetus in the 1000 mg/kg b.w had absence of tail, which were considered to be unrelated to exposure.

Body weights of the dams during the gestation period and the gestational body weight change corrected for gravid uterine weight was comparable between the control and treatment groups. No test item related changes were observed in feed consumption and in the thyroid hormone values of treatment group animals when compared with the control group animals. No gross pathological changes were observed in any of the dams of control or treatment groups. 

Uterine content examination of the dams revealed no abnormalities. The gravid uterine weight, number of corpora lutea and implantation sites, and pre-and post-implantation loses were similar between control and treatment groups. The number of early resorptions and live fetuses and the percent viable fetus per litter were comparable between the treatment and the control groups.

No soft tissue alterations were evident in the visceral examination of the fetuses. No test item related major or minor skeletal malformations were observed in the skeletal examination of fetuses. The normal skeletal variations that were observed in the fetuses of all groups were of no toxicological significance.

No test item related changes were observed in the thyroid weights and in the histopathological examination of thyroids and no changes in thyroid hormone levels were observed.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From March 07, 2011 to June 30, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

OECD 414 guideline study without any deviation. However the Certificate of Analysis is not available and the analytical verification of doses is not specified. Alkyl Ether Carboxylate is considered adequate for read-across purpose (see §"Toxicokinetics").

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan, Inc. (955, Kamibayashi, Ishioka-shi, Ibaraki Prefecture)
- Age at study initiation: no data
- Weight at study initiation: 239 to 285 g on Day 0 of gestation
- Housing: upon arrival and until paring, individually housed in in a stainless metal cage [260W × 380D × 180H (mm)]. For mating, each pair of male and female was housed together in a cage overnight for the maximum of 7 days.
- Diet (e.g. ad libitum): ad libitum, feedstuff (solid feed Labo MR Stock, lot No. 20110161, Nosan Corporation)
- Water (e.g. ad libitum): ad libitum, sterilized water ( prepared by filtration of tap water through a cartridge filter of 1-μm pore size, followed by ultraviolet light irradiation)
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): >= 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: March 10, 2011 To: April 12, 2011

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water of Japanese Pharmacopoeia grade.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor. The test substance, in an amount calculated for each dose, was weighed into a volumetric flask, and made up to volume by adding the vehicle while dissolving the test substance and swirling the solution. The dose formulations were prepared after correcting for the purity of the test substance (21.9%). That the dosing solutions were prepared at the prescribed concentrations was confirmed by the measurement of the solid content once during the study period.

VEHICLE
- Justification for use and choice of vehicle : had been prooved as appriopriate in previous studies
- Amount of vehicle: dose volume = 10 mL/kg body weight
- Lot/batch no.: lot No. 760513

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for the maximum of 7 days.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From GD6 through GD19

Frequency of treatment:

Once daily

Duration of test:

14 days

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: provided by the sponsor

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Visually inspected daily for morbidity, mortality and evidence of reaction to treatment or ill-health


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 through 20 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 3-20 (daily)
Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 0-6, 6-19 and 19-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation day 0 and 3-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weigh change intervals .

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No, not a drinking water study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross observations of organs, maternal tissues were preserved

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examination: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Soft tissue examinations (head): Yes: half per litter

Statistics:

The significant difference (significance level ≤5%) of the mean and frequency of each index parameter obtained from pregnant animals between each test substance group and the control group was calculated according to the following methods:
- Parametric data (body weight of mother animals, amount of body weight increase, food consumption, number of corpora lutea, number of implantations, number of dead embryos/fetuses, number of live fetuses, uterine weight) were subjected to Bartlett’s variance test. If the variance was uniform, data were subjected to one-way analysis of variance and, if significant difference was observed, subjected to comparison of each group to the control group by Dunnett’s test.
- Data with non-uniform variance and nonparametric data (implantation rate, embryonic/fetal mortality, sex ratio of fetuses, fetal body weight, placental weight, and data of external, visceral, and skeletal examination) were subjected to Kruskal-Wallis rank test. If a significant difference was observed, data were subjected to comparison of each group to the control group by Dunnett type test.
- Categorical data (observation for clinical signs, necropsy) were subjected to Fisher’s exact test. For the data of fetuses, the mean value obtained from litter mates was handled as that of 1 sample.

Indices:

- Postimplantation loss/litter= (No. dead fetuses, resorptions (Early/Late)/Group) / No. Gravid Females/Group.
- Summation per group (%) = Sum of postimplantation loss/litter (%) / No. litters/group.
- Postimplantation loss/litter (%) = (No. dead fetuses, resorptions (Early/Late)/litter) / No. implantation sites/litter * 100
- Viable fetuses affected/litter (%) = (No. viable fetused affected/litter) / No. viable fetuses/litter * 100

Historical control data:

No data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: mild decrease in locomotor activity in 12 of 25 animals, loose stool in 24, and soiled lower abdomen due to urine in 23 animals, on and after gestation day 7 (2 days after the start of administration); and transient mild salivation immediately after administration, on and after gestation day 6 (1 day after the start of administration). The decreased locomotor activity resolved around 4 hours after administration.
- At 60 and 250 mg/kg bw/day: no changes in clinical signs were observed (See Attached Table 7.8.2.2).

Mortality:

no mortality observed

Description (incidence):

No death was observed in any of the groups (See Attached Table 7.8.2.2).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: decreased weight gain was observed during the gestation period. A significant decrease was observed in body weight from gestation day 9 through 20 and in body weight gain during the administration period.
- At 60 and 250 mg/kg bw/day: no significant changes were observed.
- No test substance-related effects on mean body weights, body weight gains, net body weight gains or gravid uterine weights were observed (See Attached Table 7.8.2.3).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: a significant decrease in food consumption was observed from gestation day 6 through 20.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change (See Attached Table 7.8.2.4).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Animals in 1000 mg/kg bw/day group showed a significant decrease in placental weight but did not show any significant change in other test parameters.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change in uterine weight, or placental weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Necropsy revealed mild thickening of forestomach in 23 of 25 animals in 1000 mg/kg bw/day group. Mother animals in the control group, 60 mg/kg bw/day group, and 250 mg/kg bw/day group did not show gross abnormalities in the visceral organs, showing no abnormalities in the uterus, such as amniotic fluid, either (See Attached Table 7.8.2.5).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Dead fetuses:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The number of pregnant animals was 23 in the control group, 25 in 60 mg/kg bw/day group, 24 in 250 mg/kg bw/day group, and 25 in 1000 mg/kg bw/day group (See Attached Table 7.8.2.6).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Clinical signs, decreased body weight gain and food consumption observed at 1000 mg/kg bw/day

Key result

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant changes were observed in body weight of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant changes were observed in the number of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes were observed in the sex ratio in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant changes were observed in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- External examination including oral examination was conducted in 324 animals in the control group, 375 in 60 mg/kg bw/day group, 359 in 250 mg/kg bw/day group, and 360 in 1000 mg/kg bw/day group. Dwarf fetuses were observed as follows: 3 (incidence 0.9%) in the control group, 2 (0.7 %) in 60 mg/kg group, 3 (0.8 %) in 250 mg/kg bw/day group, and 1 (0.3 %) in 1000 mg/kg bw/day group. One (0.3%) each of cleft-lip fetus and tail-less fetus was observed in 1000 mg/kg bw/day group.
- There was no significant difference between the control group and each test substance group in the incidence of fetuses with external abnormality or in the incidence of each type of external abnormality (See Attached Table 7.8.2.7).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference between the control group and each test substance group in the incidence of skeletal variations (See Attached Table 7.8.2.8) .
Skeletal examination was conducted in 160 fetuses in the control group, 186 in 60 mg/kg bw/day group, 172 in 250 mg/kg bw/day group, and 193 in 1000 mg/kg bw/day group.
- Skeletal anomalies observed were fused cervical vertebral arch and fused thoracic vertebral arch in 1 fetus each, 2 in total (incidence 1.0%) in the control group. In the test substance groups, the only skeletal anomaly observed was partially missing sacral vertebral bones in 1 fetus (0.5%) in 1000 mg/kg bw/day group.
- The following skeletal variations were observed in each group: Cervical rib (incidence 0-3.4%), asymmetric sternebrae (0-0.5%), dumbbell-shaped thoracic vertebral body (1.7-5.2%), split of the thoracic vertebral body (0.3-2.3%), short thoracic vertebral arch (0-0.5%), small thoracic vertebral body (0-1.0%), short 13th rib (0-1.6%), 14th rib (0-0.6%), lumbar rib (4.5-9.8%), dumbbell-shaped lumbar vertebral body (0-0.5%), sacralization of lumbar vertebral arch (0-0.5%), variation of the number of lumbar spinal bones (0-0.5%), split of the lumbar vertebral body (0-0.5%), small lumbar vertebral body (0-0.5%), and lumbar vertebral deformity (0-0.5%).
- The number of fetuses with any of these skeletal variations (incidence) was 21 (13.8 %) in the control group, 27 (13.7 %) in 60 mg/kg bw/day group, 16 (8.9 %) in 250 mg/kg bw/day group, and 31 (17.5 %) in 1000 mg/kg bw/day group, with the incidence showing no significant difference between the control group and each test substance group or no tendency of dose-dependent increase. The incidence of each type of skeletal variation did not show any significant difference between the control group and each test substance group, nor did it show any tendency of dose-dependent increase.
- As for ossification, all parameters including the numbers of ossified sternebral bones, caudal vertebral bones, metacarpal bones, and metatarsal bones were comparable between the control group and each test substance group, showing no significant changes. In 250 mg/kg bw/day group, one fetus (incidence 0.5%) showed delayed ossification of cervical vertebral arch and 3 fetuses (1.4%) showed delayed ossification of ischial bones, but none of them were significant.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral examination was conducted on 164 fetuses in the control group, 189 in 60 mg/kg bw/day group, 187 in 250 mg/kg bw/day group, and 167 in 1000 mg/kg bw/day group (See Table 7.8.2.9)
- No fetuses with visceral anomalies were observed either in the control group or in the test substance groups.
- Visceral variations observed were thymic cervical residue (incidence 0.5%-1.3%) in all treatment groups and persistent left umbilical artery (1.7%) in 1000 mg/kg bw/day group.
- The number of fetuses with either of these visceral variations (incidence) was 2 (1.1%) in the control group, 2 (1.2%) in 60 mg/kg bw/day group, 3 (1.3%) in 250 mg/kg bw/day group, and 3 (2.1%) in 1000 mg/kg bw/day group, with no significant difference in the incidence between the control group and each test substance group. Also, no significant difference was observed in the incidence of each type of visceral variation between the control group and each test substance group.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

none

Conclusions:

Under the test conditions, the NOAEL for maternal systemic toxicity was 1000 mg /kg bw/day based on clinical signs, decreased body weight gain and food consumption observed at 1000 mg/kg bw/day that are due to the irritant potential of the test substance. The NOAEL for developmental toxicity was 1000 mg/kg bw/day as no effects were observed at all tested doses.

Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, the test substance diluted in deionized water was administered to 25 females SD [Crl: CD (SD)] rats/dose by gavage at dose levels of 0, 60, 250 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. Animals at 1000 mg/kg bw/day showed changes in clinical signs including decreased locomotor activity, loose stool, soiled lower abdomen due to urine, and transient salivation immediately after administration, as well as decreased weight gain, decreased food consumption, thickening of forestomach, and decreased placental weight. No effects were observed in 60 and 250 mg/kg bw/day groups.

There were no test article-related internal findings at the scheduled necropsy. For the effect on fetuses, no significant changes were observed in the number of live fetuses, embryo/fetal mortality, sex ratio, body weight of live fetuses, or in the external, visceral, or skeletal examination of fetuses, at all tested animals. No test article-related fetal malformations or developmental variations were noted at any dose level in this study. No other signs of developmental toxicity were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered to be the NOAEL(systemic effects) for maternal toxicity and the NOAEL for developmental toxicity.

Under the test conditions, the test substance is not classified according to Regulation (EC) No.1272/2008 (CLP) criteria.

This study is acceptable and satisfies the requirement for developmental toxicity endpoint.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See the RAAF Document.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: mild decrease in locomotor activity in 12 of 25 animals, loose stool in 24, and soiled lower abdomen due to urine in 23 animals, on and after gestation day 7 (2 days after the start of administration); and transient mild salivation immediately after administration, on and after gestation day 6 (1 day after the start of administration). The decreased locomotor activity resolved around 4 hours after administration.
- At 60 and 250 mg/kg bw/day: no changes in clinical signs were observed (See Attached Table 7.8.2.2).

Mortality:

no mortality observed

Description (incidence):

No death was observed in any of the groups (See Attached Table 7.8.2.2).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: decreased weight gain was observed during the gestation period. A significant decrease was observed in body weight from gestation day 9 through 20 and in body weight gain during the administration period.
- At 60 and 250 mg/kg bw/day: no significant changes were observed.
- No test substance-related effects on mean body weights, body weight gains, net body weight gains or gravid uterine weights were observed (See Attached Table 7.8.2.3).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 1000 mg/kg bw/day: a significant decrease in food consumption was observed from gestation day 6 through 20.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change (See Attached Table 7.8.2.4).

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Animals in 1000 mg/kg bw/day group showed a significant decrease in placental weight but did not show any significant change in other test parameters.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change in uterine weight, or placental weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Necropsy revealed mild thickening of forestomach in 23 of 25 animals in 1000 mg/kg bw/day group. Mother animals in the control group, 60 mg/kg bw/day group, and 250 mg/kg bw/day group did not show gross abnormalities in the visceral organs, showing no abnormalities in the uterus, such as amniotic fluid, either (See Attached Table 7.8.2.5).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Dead fetuses:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

See Attached Table 7.8.2.6.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

The number of pregnant animals was 23 in the control group, 25 in 60 mg/kg bw/day group, 24 in 250 mg/kg bw/day group, and 25 in 1000 mg/kg bw/day group (See Attached Table 7.8.2.6).

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Clinical signs, decreased body weight gain and food consumption observed at 1000 mg/kg bw/day with Alkyl Ether Carboxylate.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 2 400 mg/kg bw/day

Based on:

other: Trideceth-2 Carboxamide MEA

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: decreased body weight gain and food consumption observed at 1000 mg/kg bw/day

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant changes were observed in body weight of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant changes were observed in the number of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes were observed in the sex ratio in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No significant changes were observed in any of the test substance groups (See Attached Table 7.8.2.6).

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- External examination including oral examination was conducted in 324 animals in the control group, 375 in 60 mg/kg bw/day group, 359 in 250 mg/kg bw/day group, and 360 in 1000 mg/kg bw/day group. Dwarf fetuses were observed as follows: 3 (incidence 0.9%) in the control group, 2 (0.7 %) in 60 mg/kg group, 3 (0.8 %) in 250 mg/kg bw/day group, and 1 (0.3 %) in 1000 mg/kg bw/day group. One (0.3%) each of cleft-lip fetus and tail-less fetus was observed in 1000 mg/kg bw/day group.
- There was no significant difference between the control group and each test substance group in the incidence of fetuses with external abnormality or in the incidence of each type of external abnormality (See Attached Table 7.8.2.7).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was no significant difference between the control group and each test substance group in the incidence of skeletal variations (See Attached Table 7.8.2.8) .
Skeletal examination was conducted in 160 fetuses in the control group, 186 in 60 mg/kg bw/day group, 172 in 250 mg/kg bw/day group, and 193 in 1000 mg/kg bw/day group.
- Skeletal anomalies observed were fused cervical vertebral arch and fused thoracic vertebral arch in 1 fetus each, 2 in total (incidence 1.0%) in the control group. In the test substance groups, the only skeletal anomaly observed was partially missing sacral vertebral bones in 1 fetus (0.5%) in 1000 mg/kg bw/day group.
- The following skeletal variations were observed in each group: Cervical rib (incidence 0-3.4%), asymmetric sternebrae (0-0.5%), dumbbell-shaped thoracic vertebral body (1.7-5.2%), split of the thoracic vertebral body (0.3-2.3%), short thoracic vertebral arch (0-0.5%), small thoracic vertebral body (0-1.0%), short 13th rib (0-1.6%), 14th rib (0-0.6%), lumbar rib (4.5-9.8%), dumbbell-shaped lumbar vertebral body (0-0.5%), sacralization of lumbar vertebral arch (0-0.5%), variation of the number of lumbar spinal bones (0-0.5%), split of the lumbar vertebral body (0-0.5%), small lumbar vertebral body (0-0.5%), and lumbar vertebral deformity (0-0.5%).
- The number of fetuses with any of these skeletal variations (incidence) was 21 (13.8 %) in the control group, 27 (13.7 %) in 60 mg/kg bw/day group, 16 (8.9 %) in 250 mg/kg bw/day group, and 31 (17.5 %) in 1000 mg/kg bw/day group, with the incidence showing no significant difference between the control group and each test substance group or no tendency of dose-dependent increase. The incidence of each type of skeletal variation did not show any significant difference between the control group and each test substance group, nor did it show any tendency of dose-dependent increase.
- As for ossification, all parameters including the numbers of ossified sternebral bones, caudal vertebral bones, metacarpal bones, and metatarsal bones were comparable between the control group and each test substance group, showing no significant changes. In 250 mg/kg bw/day group, one fetus (incidence 0.5%) showed delayed ossification of cervical vertebral arch and 3 fetuses (1.4%) showed delayed ossification of ischial bones, but none of them were significant.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral examination was conducted on 164 fetuses in the control group, 189 in 60 mg/kg bw/day group, 187 in 250 mg/kg bw/day group, and 167 in 1000 mg/kg bw/day group (See Table 7.8.2.9)
- No fetuses with visceral anomalies were observed either in the control group or in the test substance groups.
- Visceral variations observed were thymic cervical residue (incidence 0.5%-1.3%) in all treatment groups and persistent left umbilical artery (1.7%) in 1000 mg/kg bw/day group.
- The number of fetuses with either of these visceral variations (incidence) was 2 (1.1%) in the control group, 2 (1.2%) in 60 mg/kg bw/day group, 3 (1.3%) in 250 mg/kg bw/day group, and 3 (2.1%) in 1000 mg/kg bw/day group, with no significant difference in the incidence between the control group and each test substance group. Also, no significant difference was observed in the incidence of each type of visceral variation between the control group and each test substance group.

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 2 400 mg/kg bw/day

Based on:

other: Trideceth-2 Carboxamide MEA

Sex:

male/female

Basis for effect level:

other: No effects were observed.

Abnormalities:

not specified

Developmental effects observed:

not specified

none

Conclusions:

Under the test conditions, the NOAEL for maternal toxicity (systemic effects) and the NOAEL for developmental toxicity were set to 1000 mg/kg bw/d, which corresponds to 2400 mg/kg bw/day of the registered substance.

Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, the test substance diluted in deionized water was administered to 25 females SD [Crl: CD (SD)] rats/dose by gavage at dose levels of 0, 60, 250 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. Animals at 1000 mg/kg bw/day showed changes in clinical signs including decreased locomotor activity, loose stool, soiled lower abdomen due to urine, and transient salivation immediately after administration, as well as decreased weight gain, decreased food consumption, thickening of forestomach, and decreased placental weight. No effects were observed in 60 and 250 mg/kg bw/day groups.

There were no test article-related internal findings at the scheduled necropsy. For the effect on fetuses, no significant changes were observed in the number of live fetuses, embryo/fetal mortality, sex ratio, body weight of live fetuses, or in the external, visceral, or skeletal examination of fetuses, at all tested animals. No test article-related fetal malformations or developmental variations were noted at any dose level in this study. No other signs of developmental toxicity were noted.

Based on the results of this study the dose level of 1000 mg/kg bw/day was considered to the NOAEL maternal toxicity (systemic effects) and developmental toxicity, which corresponds to 2400 mg/kg bw/d of the registered substance.
Under the test conditions, the registered substance is not classified according to Regulation (EC) No.1272/2008 (CLP) criteria.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The key study for this endpoint is Prenatal Developmental Toxicity Dhandapani P 2023. 1 (reliable without restriction)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Read-across approach - Supporting Information Only:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, the test substance (i.e. source substance) diluted in deionized water was administered to 25 females Sprague Dawley rats/dose by gavage at dose levels of 0, 60, 250 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. Animals at 1000 mg/kg bw/day showed changes in clinical signs including decreased locomotor activity, loose stool, soiled lower abdomen due to urine, and transient salivation immediately after administration, as well as decreased weight gain, decreased food consumption, thickening of forestomach, and decreased placental weight. No effects were observed in 60 and 250 mg/kg bw/day groups.

 

There were no test substance-related internal findings at the scheduled necropsy. For the effect on fetuses, no significant changes were observed in the number of live fetuses, embryo/fetal mortality, sex ratio, body weight of live fetuses, or in the external, visceral, or skeletal examination of fetuses, at all tested animals. No test article-related fetal malformations or developmental variations were noted at any dose level in this study. No other signs of developmental toxicity were noted.

 

Based on the results of this study conducted with the source substance, the dose level of 1000 mg/kg bw/day was considered to the NOAEL maternal toxicity (systemic effects) and developmental toxicity, which corresponds to 2400 mg/kg bw/d of the target substance based on the molecular weight of the main constituent (which is the source substance) and its concentration in the target substance. This constituent is considered to be the main constituent driving the potential toxicity of the target substance if some could occur (see the RAAF document).

The absence of both maternal and developmental toxicity is corroborated by the absence of toxicity observed in the screening in the reproduction/developmental screening test (OECD 421) conducted with AA 15 where no toxicity is observed in the pups following clinical and macroscopical examination.

Again, both substances have similar results profiles in an in vitro study for the developmental potential where metabolic perturbation at concentrations similar to those that impacted cell viability was observed. Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity for AA 15 and Alkyl Ether Carboxylate.

 

 

 

 

 

Toxicity to reproduction: other studies

Description of key information

Study was conducted with AA 15 and with Alkyl Ether Carboxylate (i.e. the source substance) to predict a developmental toxicity potential (Stemina Biomarker Discover, 2019).

Both substances have similar results profiles in this study and caused metabolic perturbation at concentrations similar to those that impacted cell viability. Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity for AA 15 and Alkyl Ether Carboxylate. The results for Alkyl Ether Carboxylate is corroborated by the developmental toxicity study conducted with the substance showing the absence of developmental toxicity.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 September 2019 to 05 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Study conducted to streghthen the pre-developmental study in a read-across approach: see section 7.8.2

Reason / purpose for cross-reference:

reference to same study

Conclusions:

Under the test conditions, the dTP (o/c ratio) is 13.3 µg/mL and toxicity potential (TP, cell viability) is 13.4 µg/mL. The test article elicited a biphasic response in the o/c ratio due to increased cystine uptake prior to cell death, which may be an adaptive response to oxidative stress, followed by cell death when the cells cannot overcome the stress. However, the test article caused metabolic perturbation at concentrations similar to those that impacted cell viability. Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity. and that there is no evidence for any potential for developmental toxicity.

Executive summary:

The purpose of this study was to predict a test article developmental toxicity potential. The impact of test article exposure on ornithine and cystine metabolism in human induced pluripotent stem (iPS) cells was measured for the test article and prediction of the potential for developmental toxicity was made through application of the hPS cell-based devTOXquickPredict assay. Exposure spanned a range of eight treatment levels ( 0.03 - 100 µg/mL)

The dTP (o/c ratio) is 13.3 µg/mL and toxicity potential (TP, cell viability) is 13.4 µg/mL. The test article elicited a biphasic response in the o/c ratio due to increased cystine uptake prior to cell death, which may be an adaptative response to oxidative stress, followed by cell death when the cells cannot overcome the stress. The test article caused metabolic perturbation at concentrations similar to those that impacted cell viability. Human iPS cell viability was decreased by the test article at concentrations similar to those that were cytotoxic in mouse lymphoma L5178Y cells in the absence of S9 after after 24 hours of exposure (31.3 µg/mL). Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity.

Additional information

Supporting Information Only

A study was conducted to predict the developmental toxicity potential of Alkyl Ether Carboxylate (Stemina Biomarker Discover, 2019). The impact of test substance exposure on ornithine and cystine metabolism in human induced pluripotent stem (iPS) cells was measured for the test article and prediction of the potential for developmental toxicity was made through application of the hPS cell-based devTOXquickPredict assay. Exposure spanned a range of eight treatment levels ( 0.1 - 300 µg/mL).

The dTP (o/c ratio) is 54.1 µg/mL and toxicity potential (TP, cell viability) is 80.1 µg/mL. The test article elicited a biphasic response in the o/c ratio due to increased cystine uptake prior to cell death, which may be an adaptative response to oxidative stress, followed by cell death when the cells cannot overcome the stress. The test article caused metabolic perturbation at concentrations similar to those that impacted cell viability. Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity.

 

In the same study above, the purpose was to predict a developmental toxicity potential of AA 15. The impact of test article exposure on ornithine and cystine metabolism in human induced pluripotent stem (iPS) cells was measured for the test article and prediction of the potential for developmental toxicity was made through application of the hPS cell-based devTOXquickPredict assay. Exposure spanned a range of eight treatment levels ( 0.03 - 100 µg/mL)

The dTP (o/c ratio) is 13.3 µg/mL and toxicity potential (TP, cell viability) is 13.4 µg/mL. The test article elicited a biphasic response in the o/c ratio due to increased cystine uptake prior to cell death, which may be an adaptative response to oxidative stress, followed by cell death when the cells cannot overcome the stress. The test article caused metabolic perturbation at concentrations similar to those that impacted cell viability. Human iPS cell viability was decreased by the test article at concentrations similar to those that were cytotoxic in mouse lymphoma L5178Y cells in the absence of S9 after after 24 hours of exposure (31.3 µg/mL). Therefore, it can be concluded that the metabolic disturbance was a consequence of cytotoxicity and that there is no evidence for any potential for developmental toxicity.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed according to the Regulation (EC) No 1272/2008 and to the GHS.