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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The oral administration of registered substance, Pigment Brown 41, over the time of the study, carried out according to the OECD guideline 421 (for males with a total of 28 days and for females ranging from 49 to 60 days) did not produce any indication of systemic, reproduction and developmental toxicity in any of the tested dose levels i.e., 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

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 21 March 2022 to 25 November 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN

- Premating exposure duration for animals: 2 Weeks
- Basis for dose level selection: The doses of 0, 111, 333 and 1000 mg/kg body weight/day was selected as vehicle control, low, mid and high dose levels based on toxicity information on similar pigments which had no effects when administered orally to the rats.
- Termination time for F1: Postnatal day 13
- Route of administration: Oral
- choice of species and strain: Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
- vehicle: 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL. Carboxymethyl cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies
- number of animals: 96 (48 Males + 48 Females) as per guideline
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd,
Charles River Technology Licensee

- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: Males: 234.09 to 297.15 g, Females: 210.56 to 240.29 g
- Housing: During acclimatization - two animals, Pre-mating - two animals, Mating - two animals, Post-mating : Males were housed two, while females were housed individually
- Diet : Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum
- Water : Deep bore-well water passed through reverse osmosis unit and was provided ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 24.7
- Humidity (%): 44 to 66
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 01 April 2022 To: 19 June 2022
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v Carboxymethyl cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of the test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension is formed. After that, the entire quantity of the formulation was transferred into a measuring cylinder. A small amount of vehicle was added to rinse the mortar, and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated (many times) to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to the required quantity with the vehicle to get the desired concentration of 11, 33 and 100 mg/mL of test item for low, mid and high dose groups, respectively.

VEHICLE
- Justification for use and choice of vehicle : 0.5% w/v Carboxymethyl cellulose, Carboxymethyl cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 0 mg/mL
- Amount of vehicle (if gavage): 10mL/Kg
- Batch no. : SLCH1520
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 12 days
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations were performed during week 1 and week 5 of the treatment. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was less than 10%.
Duration of treatment / exposure:
Males: The males were treated for two weeks pre-mating, during mating period and up to the day before sacrifice (total of 28 days of dose administration).
Females:
- The pregnant animals were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 49 to 60 days).
- The non-pregnant animals were treated during two-week pre-mating period, during mating until confirmed as mated and further 26 days from the day of confirmation of mating
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 to 14 weeks
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The doses of 0, 111, 333 and 1000 mg/kg body weight/day was selected as vehicle control, low, mid and high dose levels based on toxicity information on similar pigments which had no effects when administered orally to the rats.
- Rationale for animal assignment : The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study does not exceed ±20% (Males: -11.55 to 10.81%; Females: -10.06 to 5.56%) of the mean body weight of each sex.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: The males were weighed
- at the receipt
- on the day of randomization and grouping
- on the first day of dosing, weekly thereafter
- and at termination.
Females:
- The females were weighed
- at receipt
- on the day of randomization and grouping
- on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating.
- All the pregnant animals were weighed on gestation day (GD) 0, 7, 14, and 20 and on lactation day (LD) 1, 4, 7, 13 and 14 (fasting/terminal body weight).
- The non-pregnant animals were weighed weekly once until termination and the fasting/terminal body weights were also recorded before scheduled sacrifice.


OTHER: feed consumption
Males: Cage wise feed consumption was measured for all males once in a week during pre-mating period coinciding with body weight recording.
Females:
- Cage wise feed consumption was measured for all females once in a week during premating period coinciding with body weight recording.
- Cage wise feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13.
- Cage wise feed consumption was measured for all non-pregnant animals once in a week coinciding with body weight recording.
Oestrous cyclicity (parental animals):
Oestrus cyclicity was monitored during
- two weeks after five days of acclimatization to screen the females for regular oestrus cyclicity
- two week pre-mating treatment period
- during mating treatment period until confirmation of mating
- at termination
Sperm parameters (parental animals):
Parameters examined in adult male (parents)generations:testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, however one/ two pups were eliminated if more than 10 pups/litter for measurement of serum total T4 levels

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males were sacrificed after completion of 28 days of treatment
- Maternal animals: All the littered females were sacrificed on lactation day 14. The non-pregnant females were sacrificed after 26 days from the last day of confirmation of mating.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues Epididymides, Prostate, Seminal vesicles and coagulating glands, Testes, Ovaries , Uterus with cervix and Thyroid along with parathyroid#(post fixation) were weighed,A detailed histopathological examination was performed on the ovaries, testes, vagina and epididymides from the high dose and the control group animals A detailed histopathological examination was also performed on ovaries, vagina and uterus with cervix for one moribund sacrificed female.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and with particular attention to the external reproductive genitals and the findings were recorded.
Statistics:
Parametric - One-way ANOVA with Dunnett’s post test:
• Body weight (weekly/gestation/lactation)
• Percent change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum T4 values (adults/pups)

Non-parametric - Kruskal-Wallis.
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during the lactation period
• Litter size at birth and during the lactation period
• No. of resorptions/litter
• Post-implantation loss/litter
• Postnatal loss/litter
Cross Tabs - Chi-square test.
• Pregnancy Index (%)
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions
Reproductive indices:
Male and Female reproductive indices
Male mating index (%) = No. of males with confirmed mating / Total no. of males cohabited X 100
Female mating index (%) = No. of sperm-positive females / Total no. of females cohabited X 100
Male fertility index (%) = No. of males impregnating a female / Total no. of males mated X 100
Female fertility index (%) = No. of pregnant females / No. of sperm-positive females X 100
Gestation index (%) = No. of females with live born / No. of females with evidence of pregnancy X 100
Parturition index (%) = No. of females littered / No. of females with evidence of pregnancy X 100
Pregnancy index (%) = No. of pregnant females / No. of females with confirmed mating X 100
Post-implantatin loss (%) = No. of implantations - No. of viable pups / No. of implantations X 100
Total postnatal loss (%) = Total no. of pups dead/cannibalized during postnatal period / Total no. of live pups delivered X 100
Offspring viability indices:
Live birth index (%) per litter = No. of pups born alive / Total no. of pups born X 100
Pup survival index (%) on LD 4/7/13 = Total No. of live pups on (LD 4/7/13) / No. of pups born/4/7 X 100
Anogenital distance (AGD) ratio = Anogenital distance (mm) / Cube root of PND 4 pup weight X 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the animals of both sexes from all the tested dose groups and vehicle control group throughout the experimental period.
All animals from group G3 & G4 were noted with slight brown coloured faecal matter from treatment day 2 onwards.
However, 1 out of 12 females from group G4 was noted with dystocia, lethargy and wet perineum during pregnancy and subjected to moribund sacrifice on gestation day 23. This noted observation is considered as incidental but not due to test item exposure as there were no such incidences noted in any of the other littered females from the same dose level. Also, the noted frequency of dystocia is within the in-house historical control range.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in mean feed consumption in all the tested dose groups of both sexes when compared with the vehicle control group. The noted statistically significant reduction in mean feed consumption during week 1 and 2 in group G3 females when compared with the vehicle control group is considered as incidental and un-related to test item exposure as the reduction was not occurred in dose dependent manner and also no changes noted in mean body weight during the similar period at this dose level.
There were no effects noted in mean gestational feed consumption in any of the tested dose groups when compared with vehicle control group throughout gestation period.
There were no effects noted in mean lactation feed consumption in any of the tested dose groups when compared with vehicle control group throughout lactation period.
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
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no effects or changes noted in mean serum T4 levels in any of the tested dose group adult males when compared with the vehicle control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related, microscopic findings noted in any of the animals of both sexes from high dose group. However, one female from group G4, which was subjected to moribund sacrifice, was noted with haemorrhage in vagina. This noted observation can be due to dystocia, but not due to any test item exposure
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related irregularities observed in oestrus cyclicity of females from any of the tested dose groups during pre-mating and mating treatment periods. However, some of the animals from all the tested dose groups and vehicle control group shown occasional irregular oestrus cycle during treatment period. These noted irregularities in duration of oestrus cyclicity in all the tested dose groups are considered as incidental and unrelated to treatment as the same females were noted with normal preceding oestrus cycles and there were no effects noted in reproductive performance of these females
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
a. Male mating index
All the males, 12 (out of 12) from each test dose group and vehicle control group were confirmed with mating during cohabitation period with a mating index of 100.00%.
b. Male fertility index
A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 11 (out of 12) mated males were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 83.33%, 91.67%, 83.33% and 91.67% from the dose groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with the vehicle control group.
c. Female mating index
All the females, 12 (out of 12) from each test dose group and vehicle control group were confirmed as sperm positive during vaginal smear examination with mating during cohabitation period with a mating index of 100.00%.
d. Female fertility index
A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 11 (out of 12) sperm positive females were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 83.33%, 91.67%, 83.33% and 91.67% from dose groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with the vehicle control group.
e. Pre-coital interval/Copulatory interval [mean time of mating]
A total of 12 pairs were left for cohabitation initially from each group. The mean
pre-coital interval was 2.75, 2.42, 3.50 and 3.50 days for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for mean
pre-coital interval in any of the tested dose groups when compared with the vehicle control group.
f. Gestation length/Duration of pregnancy
The mean gestation length [day of confirmed as successfully mated to, day of parturition] was 22.70, 22.64, 22.80 and 22.50 days for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control group.
g. Fecundity or Pregnancy index
A total of 10 (out of 12), 11 (out of 12), 10 (out of 12) and 11 (out of 12), sperm positive females were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 83.33%, 91.67%, 83.33% and 91.67% from groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for fecundity index in any of the tested dose groups when compared with the vehicle control group.
h. Gestation index
A total of 10 (out of 10), 11 (out of 11), 10 (out of 10) and 10 (out of 11) females with evidence of pregnancy were confirmed with live born pups with a gestation index of 100.00%, 100.00%, 100.00% and 90.91% from groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for gestation index in any of the tested dose groups when compared with the vehicle control group.
In group G4, 1 out of 11 pregnant females achieved pregnancy but lost complete litter due to dystocia on gestation day 23. This noted observation is considered as incidental and un-related to treatment as there were no maternal effects noted in any of the other females from the same dose level.
i. Parturition index
A total of 10 (out of 10), 11 (out of 11), 10 (out of 10) and 10 (out of 11) females with evidence of pregnancy were confirmed with parturition with a parturition index of 100.00%, 100.00%, 100.00% and 90.91% from groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for parturition index in any of the tested dose groups when compared with the vehicle control group.
j. Implantation sites and Viable pups
A mean number of 7.80, 9.55, 9.70 and 8.80 implantation sites and mean number of 7.30, 9.18, 9.70 and 8.40 viable pups were noted from groups G1, G2, G3 and G4 respectively.
There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.
k. Post-implantation loss
A mean number of 0.50, 0.36, 0.00 and 0.40 post-implantation losses with a percentage of 5.54%, 4.04%, 0.00% and 4.11% were noted from groups G1, G2, G3 and G4 respectively.
There were no statistically significant changes as for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group.
l. Postnatal loss
A mean number of 0.00, 0.00, 0.00 and 0.20 postnatal losses with a percentage of 0.00, 0.00, 0.00 and 1.67 were noted from groups G1, G2, G3 and G4 respectively.
There were no statistically significant changes noted for mean number and percentage of occurred postnatal losses in any of the tested dose groups when compared with the vehicle control group.
There were no test item related clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups, except a single occurrence of moribundity from group G4, where the dam was observed with lethargy, dystocia, and wet perineum. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all the tested dose groups of both sexes. The estimated serum T4 levels did not reveal any test item related changes of the tested dose groups. The absolute and relative organ weights of both sexes did not reveal any test item related changes from all the tested dose groups. No gross pathological changes noted in any of the animals of both sexes from all the tested dose groups during necropsy. The moribund sacrificed dam from group G4 was noted with internal gross pathological changes such as Uterus, vagina, cervix – discoloration, red, lumen with underdeveloped fetus with resorptions during gross pathological examination. No treatment related microscopic findings noted in any of the tissues/organs subjected for histopathological evaluation from high dose group animals of both sexes, except minimal haemorrhage in vagina in moribund sacrificed animal.
There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups.
No effects were noted for live birth index and pup survival index in all the tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no test item-related mortalities noted in pups from any of the test item administered groups at birth and during the post-natal period. However, on post-natal day 2, two female pups from G4 group were found dead. These incidences lacked dose correlation and gross findings and hence considered as incidental findings without any relation to administration of test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects or changes noted in mean pup [both male and female] weight per litter in any of the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7 and 13.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no occurrences or evidence of retention of nipples in any of the male pups examined on the postnatal day 13 from all the tested dose groups and vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean serum T4 hormone levels of postnatal day 13 pups estimated on litter basis in any of the tested dose group litters when compared with vehicle control group litters.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There were no test item-related developmental, external or behavioural changes and
no test item-related mortalities noted during postnatal period in any of pups from all the tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all the tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all the tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all the tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
no
Conclusions:
In conclusion, the oral administration of test item over the time of the study, carried out according to the OECD guideline 421 (for males with a total of 28 days and for females ranging from 49 to 60 days) did not produce any indication of systemic, reproduction and developmental toxicity in any of the tested dose levels i.e., 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.
Executive summary:

The objective of this Reproduction/ Developmental Toxicity Screening Test of the test item by Oral Gavage in Sprague Dawley rats was to determine the possible health hazards likely to arise from repeated exposure to the test item over a relatively limited period of time. This study was also conducted to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition and to estimate the No Observed Adverse Effect Level (NOAEL).


A total of 96 (48 males + 48 females) Sprague Dawley rats were selected for the study and distributed to four groups (G1, G2, G3 and G4). Each group consisted of 12 males and 12 females. The animals in group G1 were administered with vehicle [0.5% w/v Carboxymethyl cellulose], animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight/day for low, mid and high dose groups, respectively. Vehicle and test item formulations were administered orally by gavage with the dose volume of 10 mL/kg body weight/day once daily at similar times on each day for 7 days a week.


The males from all groups were administered with test item (G2, G3 and G4) or vehicle during (G1) during pre-mating (14-days), during mating and during post-mating period (total of 28 days). The females from all groups were administered with vehicle/test item during pre-mating (14-days), during mating, pregnancy (gestation) and up to lactation day 13. The non-pregnant females were administered with vehicle/test item during pre-mating (14-days), during mating until confirmed as mated and further 26 days from the day of confirmation of mating.


For systemic toxicity assessment, all the animals of both sexes were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity. Body weight of all the animals of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day
(GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation. Body weight of all non-pregnant females was recorded once in a week until termination. Feed consumption for all the animals of both sexes were recorded once in a week during pre-mating. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13. Serum thyroxine hormone (T4) levels were estimated for all group males by ELISA method. The organs were collected and weighed on the day of termination for all animals of both sexes and organ weight relative to terminal body weight was calculated. All the animals of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. Histopathological examination was conducted on the tissues collected from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis in the male gonads.


For reproductive toxicity assessment, all the males were evaluated for reproductive performances such as, mating and fertility indices. All the females were evaluated for reproductive performances such as, mating, fertility, gestation and parturition indices. The females were also evaluated for pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during pre-mating period and the vaginal smear examination was continued until evidence of mating. At birth (delivery) parameters such as, number of live/dead pups born, litter size, sex ratio (m/f) and live birth index per litter were observed / calculated. The litter observations during lactation period such as, number of survived or dead pups, sex ratio (m/f) and pup survival index per litter were observed / calculated. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.


For developmental toxicity assessment, all surviving pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination [postnatal day (PND) 13]. All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for anogenital distance on PND 4. All male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected to estimation of thyroxine hormone (T4) levels using ELISA method.


There were no test item related clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups, except a single occurrence of moribundity from group G4, where the dam was observed with lethargy, dystocia, and wet perineum. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all the tested dose groups of both sexes. The estimated serum T4 levels did not reveal any test item related changes of the tested dose groups. The absolute and relative organ weights of both sexes did not reveal any test item related changes from all the tested dose groups. No gross pathological changes noted in any of the animals of both sexes from all the tested dose groups during necropsy. The moribund sacrificed dam from group G4 was noted with internal gross pathological changes such as Uterus, vagina, cervix – discoloration, red, lumen with underdeveloped fetus with resorptions during gross pathological examination. No treatment related microscopic findings noted in any of the tissues/organs subjected for histopathological evaluation from high dose group animals of both sexes, except minimal haemorrhage in vagina in moribund sacrificed animal.


There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups.
No effects were noted for live birth index and pup survival index in all the tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.


There were no test item-related developmental, external or behavioural changes and
no test item-related mortalities noted during postnatal period in any of pups from all the tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all the tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all the tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all the tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.

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:
reliable and valid
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information
The oral administration of registered substance, Pigment Brown 41, over the time of the study, carried out according to the OECD guideline 421 (for males with a total of 28 days and for females ranging from 49 to 60 days) did not produce any indication of systemic, reproduction and developmental toxicity in any of the tested dose levels i.e., 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.
Effect on developmental toxicity: 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:
reliable and valid
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008


The available screening studies in combination with toxicokinetic considerations is reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for reproductive or developmental toxicity under Regulation (EC) No. 1272/2008.

Additional information