COASOL 290 PLUS

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no data available on the reproductive toxicity of the reaction mass of dibutyl esters of adipic and glutaric acid. However data exist for the methyl esters of adipic, glutaric and succinic acids, and for the dibutyl adipate. Dosing of the dibutyl esters will result in the release of the acids and butanol, therefore read-across to the dimethyl esters is considered apropriate since the major hydrolysis product of the dimethyl esters is the acids. Further information on the read-across justification is provided in the document in section 13 and section 0.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

Principles of method if other than guideline:

not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center of Charles River Laboratories Japan, Inc.
- Age at study initiation: 7 weeks of age when purchased
- Weight at study initiation: 199.5-243.4 g for males and 270.6-326.5 g for females
- Housing: The animals were housed individually in metal cages (22 × 27 × 19 cm). For dams after Day 18 of pregnancy, a stainless steel floorboard was placed on each cage floor and wood chips (White flake®, Charles River Laboratories Japan, Inc.) were provided appropriately as a bedding material.
- Diet and water (ad libitum): The animals were allowed free access to pelleted feed (CA-1, CLEA Japan, Inc.) and tap water (supplied by Hadano City Waterworks Bureau).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1°C
- Humidity (%): 55 ± 5%
- Air changes (per hr): approximately 15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in corn oil (Lot No. V4K5052, Nacalai Tesque, Inc.) and the concentrations of all solutions were adjusted to provide a constant dose volume of 5 ml/kg at each dose level. Thus prepared solutions were used for dosing. The 0.1% and 20% formulations of DBA have been confirmed to be stable for at least 8 days at room temperature in a stability test conducted by Laboratory of Analytical Chemistry, Hatano Research Institute. In addition, the content of the test substance in these dosing formulations was analyzed for each dose level during the study period (Dec. 6, 1994) and was confirmed to be within 98.3-104% of the specified concentrations

Details on mating procedure:

Females were co-housed with males in the same group on a one-to-one basis all day from the early evening on Day 15 of treatment for 2 weeks at the longest. Copulation was checked every morning by the presence/absence of a vaginal plug in the vagina or sperm in the vaginal smears. The day on which evidence of copulation was confirmed was designated as Day 0 of pregnancy. Females in which copulation was confirmed were separated from the males and housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The 0.1% and 20% formulations of DBA have been confirmed to be stable for at least 8 days at room temperature in a stability test conducted by Laboratory of Analytical Chemistry, Hatano Research Institute. In addition, the content of the test substance in these dosing formulations was analyzed for each dose level during the study period (Dec. 6, 1994) and was confirmed to be within 98.3-104% of the specified concentrations.

Duration of treatment / exposure:

Dibutyl adipate (DBA) was administered orally to male and female Sprague-Dawley (Crj:CD) rats at dose levels of 0 (corn oil, 5 ml/kg/day), 10, 300 and 1000 mg/kg from prior to mating, throughout the mating period, until 2 weeks after termination of the mating period for males and from prior to mating, throughout the mating and pregnant periods, until Day 3 of lactation for females to evaluate the potential adverse effects of DBA on reproductive function of parental animals, and development and growth of the subsequent generation in rats.

Frequency of treatment:

The dosing formulations were administered once daily, basically between 13:00 and 15:00, using gastric tubes for rats.

Details on study schedule:

Dibutyl adipate (DBA) was administered orally to male and female Sprague-Dawley (Crj:CD) rats at dose levels of 0 (corn oil, 5 ml/kg/day), 10, 300 and 1000 mg/kg from prior to mating, throughout the mating period, until 2 weeks after termination of the mating period for males and from prior to mating, throughout the mating and pregnant periods, until Day 3 of lactation for females to evaluate the potential adverse effects of DBA on reproductive function of parental animals, and development and growth of the subsequent generation in rats.

Remarks:

Doses / Concentrations:
0 mg/kg (Control)
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

4 groups of 13 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a preliminary study, since no appreciable toxic signs were observed in any female rat when DBA was administered orally for 14 days at dose levels of 500 and 1000 mg/kg, 1000 mg/kg was selected as the high dose level in consideration of the dose level of a limiting test stipulated in the OECD Guideline for the Testing of Chemicals and 300 and 100 mg/kg were selected as the mid and low dose levels, respectively, using a common ratio of approximately 3. Control animals received corn oil, the vehicle for DBA.
- Rationale for animal assignment: The animals were distributed into 4 groups of 13 animals/sex/group using a stratified randomization procedure based on the body weights measured prior to dosing on the first day of treatment (Day 1 of treatment).

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- The animals were observed for clinical signs at least once daily during the rearing period. Especially, clinical observations were conducted twice daily (prior to and after treatment) during the treatment period.

BODY WEIGHT: Yes
- Body weights were measured once weekly during the treatment period (Days 1, 8, 15, 22, 29, 36 and 43 of treatment for males and Days 1, 8 and 15 of treatment for females), and on Days 0, 7, 14 and 20 of pregnancy for the females in which copulation was confirmed and on 0 and 4 days after delivery (Days 0 and 4 of lactation, respectively) for the females which delivered.

FOOD CONSUMPTION: Yes
- Food consumption was measured once weekly on the same days as body weight measurements during the treatment period, excluding the mating period, and weekly food consumption was calculated. In addition, food consumption during Days 0-7, 7-14 and 14-20 of pregnancy for the females in which copulation was confirmed and during Days 0-4 of lactation for the females which delivered was measured.

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

not applicable

Litter observations:

All females in which copulation was confirmed were allowed to deliver naturally in all groups. For the dams in which observation of delivery was feasible, the presence/absence of abnormalities was observed during delivery. For those in which delivery could not be observed directly, the presence/absence of delivery disturbance was judged and recorded based on the symptoms after delivery. Delivery was checked between 9:00 and 11:00. For the dams in which completion of delivery was confirmed, that day was designated as the day of delivery (Day 0 of lactation) and the gestation length in days (from Day 0 of pregnancy until the day of delivery) was counted. Nursing behaviors of dams were observed daily after delivery.

The total number of pups (live + dead pups) was determined on Day 0 of lactation to calculate the delivery index [(No. of pups born / No. of implantation scars) × 100] and the birth index of live pups [(No. of live pups / No. of implantation scars) × 100]. The live pups were sexed and observed for the presence/absence of external malformations. The number of dead pups was counted daily on Days 0 to 4 of lactation to calculate the live birth index [(No. of live pups / No. of pups born) × 100] and the viability index of live pups [(No. of live pups on Day 4 of lactation / No. of live pups on Day 0 of lactation × 100]. Dead pups were necropsied and were fixed and preserved in ethanol after removal of the organs in the thoracic and abdominal cavities.

Total body weights of pups were measured separately for males and females in each litter on Days 0 and 4 of lactation and the mean body weights of pups were calculated.

Postmortem examinations (parental animals):

Males were sacrificed by exsanguination under pentobarbital anesthesia and necropsied on the day following Day 42 of treatment. The kidneys, spleen, testes and epididymides were removed and weighed. In addition, the testes and epididymides were fixed in Bouin’s solution and those from the high dose and control groups were examined histopathologically. The kidneys, spleen and the organs with gross lesions were fixed and preserved in 10% formalin.

The females which delivered and those in which copulation was confirmed but delivery was not observed were sacrificed by exsanguination under pentobarbital anesthesia and necropsied on Day 4 of lactation and the day corresponding to Day 25 of pregnancy, respectively. The kidneys and spleen were removed and weighed. The ovaries and uterus were also removed. The number of corpora lutea was counted in the ovaries under a stereoscopic microscope, and the number of implantation scars was determined in the uterus using the modified Salewski method to calculate the implantation index [(No. of implantation scars / No. of corpora lutea) × 100]. The ovaries from non-pregnant animals were fixed in Bouin’s solution and examined histopathologically. The kidneys, spleen and the organs with gross lesions were fixed and preserved in 10% formalin.

Postmortem examinations (offspring):

The pups were sacrificed with ether on Day 4 of lactation and necropsied. The organs in the thoracic and abdominal cavities were removed in block, and were fixed and preserved in 10% formalin for each litter. The carcasses were fixed and preserved in ethanol for each litter.

Statistics:

Data were analyzed for homogeneity of variance using Bartlett’s test. Individual data or litter mean served as the unit of analysis. When the variance was judged to be homogeneous, one-way analysis of variance was conducted between the groups. When significance of variance was noted between the groups, mean group values were compared between the control and each treated group using Dunnett’s or Scheffé’s test depending on whether the number of animals in each group was the same or not. When the variance was heterogeneous between the groups or zero in some groups, Kruskal-Wallis’s rank test was conducted.
When significance was noted between the groups, a multiple comparison test of the Dunnett or Scheffé type was conducted. Differences from the control group were evaluated at 5% and 1% levels of significance.

Reproductive indices:

Based on the results of mating and success/failure of pregnancy, the copulation index [(Number of copulated pairs / Number of mated pairs) × 100], fertility index [(Number of pregnant animals / Number of copulated pairs) × 100], number of days from the first day of co-housing until the day of copulation and number of recurrent vaginal estrus during the mating period were calculated.
The mean gestation length in days and the gestation index [(Number of females with live pups / Number of pregnant females) × 100] were calculated for each group. The number of corpora lutea was counted in the ovaries under a stereoscopic microscope, and the number of implantation scars was determined in the uterus using the modified Salewski method to calculate the implantation index [(No. of implantation scars / No. of corpora lutea) × 100].

Offspring viability indices:

The total number of pups (live + dead pups) was determined on Day 0 of lactation to calculate the delivery index [(No. of pups born / No. of implantation scars) × 100] and the birth index of live pups [(No. of live pups / No. of implantation scars) × 100]. The live pups were sexed and observed for the presence/absence of external malformations. The number of dead pups was counted daily on Days 0 to 4 of lactation to calculate the live birth index [(No. of live pups / No. of pups born) × 100] and the viability index of live pups [(No. of live pups on Day 4 of lactation / No. of live pups on Day 0 of lactation × 100]. Dead pups were necropsied and were fixed and preserved in ethanol after removal of the organs in the thoracic and abdominal cavities.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) - No death occurred in any group. Transient salivation was observed after dosing in all DBA treated groups from Day 2 of treatment.The number of animals that exhibited salivation increased dose-dependently as follows: 3, 11 and 13 males and 2, 9 and 13 females in the 100, 300 and 1000 mg/kg groups, respectively. No other clinical signs were observed in any animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) - Actual body weighs decreased slightly in the 1000 mg/kg male group, but no statistically
significant differences were noted between the control and each DBA treated group. In the female animals, no statistically significant differences were noted between the control and each DBA treated group in body weights prior to mating, during the pregnant period or after delivery. No statistically significant differences were noted between the control and each DBA treated group in the food consumption during the treatment period fro males and prior to mating, during the pregnant period or after delivery, for the female animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) - There were no differences between the control and each DBA treated groups in the copulation or fertility index. In addition, no statistically significant differences were noted between the control and each DBA treated group in the pairing days until copulation or the number of vaginal estrus on the paring days.

ORGAN WEIGHTS (PARENTAL ANIMALS) -
Males - Statistically significant increases were noted in the relative weights of the kidneys (p < 0.05) in the 1000 mg/kg group and the spleen (p < 0.05, p < 0.01) in the 100 and 1000 mg/kg groups. However, no statistically significant differences from the control group were noted in weight of the testes or epididymides in any DBA treated group.
Females - A slight increase in the relative weight of the kidneys in the 1000 mg/kg group and a statistically significant increase in the absolute weight of the spleen (p < 0.05) in the 100 mg/kg group were noted. However, no statistically significant differences from the control group were noted in the relative weight of the spleen in any DBA treated group.

GROSS PATHOLOGY (PARENTAL ANIMALS) -
Males - Gross lesions observed in the lungs included: a dark reddish or reddish spot in 2 animals in the 300 mg/kg group and in 1 animal in the 1000 mg/kg group; dark reddish or reddish area in 1 animal in the 100 mg/kg group and in 2 animals each in the 300 and 1000 mg/kg groups; and a pale colored or grayish white spot in 1 animal each in the 100 and 1000 mg/kg groups. In the kidneys, dilatation of the renal pelvis was observed in 1 animal each in the control and 300 mg/kg groups and in 2 animals each in the 100 and 1000 mg/kg groups and a recessed area was observed in 1 animal each in the 100 and 300 mg/kg groups. In addition, the following gross lesions were observed in 1 animal each: diaphragmatic hernia of the liver in the 100 mg/kg group; dark discoloration of the liver in the 1000 mg/kg group; enlargement of the spleen in the 100 mg/kg group; a white spot in the spleen in the 1000 mg/kg group; diverticulum in the jejunum in the 300 mg/kg group; and a yellowish white nodule in the epididymis in the control group. However, the incidences of all lesions were not dose-dependent.
Females - Although a dark reddish area in the lung was observed in 2 control animals and dark reddish discoloration and a dark reddish spot in the lung and a forestomach ulcer were observed in 1 animal each in the 300 mg/kg group, no gross lesions were observed in any animal in the 100 or 1000 mg/kg group.

HISTOPATHOLOGY (PARENTAL ANIMALS) -
Males - In the testes, a decreased number of germ cells and focal atrophy of only a few seminiferous tubules were noted in 1 control animal. In the epididymides, spermatic granuloma in 1 control animal and very slight infiltration of lymphocytes in 5 control animals and 3 animals in the 1000 mg/kg group were noted. However, no histopathological lesions attributable to treatment with DBA were evident.
Females - No histopathological lesions were evident in the ovaries of any non-pregnant animal that was 1 each in the control and all DBA treated groups.

OTHER FINDINGS (PARENTAL ANIMALS) -
Reproductive performance - There were no differences between the control and each DBA treated groups in the copulation or fertility index. In addition, no statistically significant differences were noted between the control and each DBA treated group in the pairing days until copulation or the number of vaginal estrus on the paring days.
Gestation index and gestation length in days - The gestation index was 100% in all groups. No statistically significant differences were noted in the gestation length in days between the control and each DBA treated group.
Number of corpora lutea and implantation scars and implantation index - No statistically significant differences were noted between the control and each DBA treated group in the number of corpora lutea or implantation scars or in the implantation index of pregnant female.
Delivery and nursing behaviours - Among the dams in which delivery observation was feasible (8/12, 4/12, 7/12 and 8/12 dams in the control, 100, 300 and 1000 mg/kg groups, respectively), abnormalities in delivery (prolonged delivery time, no gathering of pups) were noted in 1 dam in the 1000 mg/kg group. Among the dams in which delivery could not be observed at that moment, changes judged to be abnormalities in delivery (no gathering of pups, soiled fur in the abdominal region) were observed in 1 dam in the 300 mg/kg group. However, no abnormal delivery attributable to treatment with DBA was evident in any dam.
One dam in the 300 mg/kg group whose delivery was judged to be abnormal exhibited poor nursing behavior (scattering of pups, no milk in the stomach of pups and subnormal body surface temperature of pups) from Day 0 of lactation and all pups died by Day 2 of lactation. No abnormal nursing behavior was observed in the other dams.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slight effect on male bodyweight only

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: for reproduction function of parental animals

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING) - Although a statistically significant decrease (p < 0.05) in the viability index of live pups was noted in the 1000 mg/kg group as compared to the control group, no statistically significant differences were noted between the control and each DBA treated group in the delivery, live birth or birth index or sex ratio.

BODY WEIGHT (OFFSPRING) - Slightly decreased body weights of pups were noted in the 1000 mg/kg group on Days 0 and 4 of lactation, but no statistically significant differences were noted between the control and each DBA treated group.

GROSS PATHOLOGY (OFFSPRING) - In the external observation of live pups on Day 0 of lactation, no malformations were observed. In the morphological observation of dead pups, only a short tail was observed in 1 pup in the 1000 mg/kg group. In addition, necropsy on Day 4 of lactation revealed agenesis of the midgut in 1 pup in the 100 mg/kg group. However, no malformations attributable to treatment with DBA were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

None

Conclusions:

Based on these results, the no observed adverse effect levels (NOAELs) of dibutyl adipate (DBA) were considered to be 300 mg/kg for general toxicity of parental animals and for the subsequent generation and 1000 mg/kg for reproductive function of parental animals.

Executive summary:

Dibutyl adipate (DBA) was administered orally to male and female Sprague-Dawley (Crj:CD) rats at dose levels of 0 (corn oil, 5 ml/kg/day), 10, 300 and 1000 mg/kg from prior to mating, throughout the mating period, until 2 weeks after termination of the mating period for males and from prior to mating, throughout the mating and pregnant periods, until Day 3 of lactation for females to evaluate the potential adverse effects of DBA on reproductive function of parental animals, and development and growth of the subsequent generation in rats. The results of this study are summarized as follows:

Parental findings - No death occurred in any treated group. Clinical signs considered to be toxic effects were not evident. Body weights were slightly decreased in males in the 1000 mg/kg group, but no appreciable differences were noted in body weights of females or food consumption in males or females between the control and each DBA treated group. Gross pathology and histopathology of the internal genital organs revealed no lesions attributable to DBA treatment. The terminal kidney weights were increased in males and females in the 1000 mg/kg group.

Reproductive function findings - No abnormalities attributable to DBA treatment were noted in the copulation or fertility index in males or females, or maintenance of pregnancy, delivery or nursing behavior of females.

Findings in pups - In the 1000 mg/kg group, the viability index of live pups on Day 4 of lactation was decreased and the body weights on Days 0 and 4 of lactation were slightly decreased. However, no malformations suggestive of treatment-related effects were observed in any DBA treated group.

Based on these results, the no observed adverse effect levels (NOAELs) of DBA were considered to be 300 mg/kg for general toxicity of parental animals and for the subsequent generation and 1000 mg/kg for reproductive function of parental animals.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

not specified

Principles of method if other than guideline:

- Inhalation exposure.
- Male rats were also exposed until end of lactation period.
- Female rats were not exposed from gestation day 19 through postpartum day 3.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kingston, NY;
- Age at study initiation: (P) 4 wks
- Housing: individually, except during mating (1 male, 1 female)
- Diet (e.g. ad libitum): a libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: inhalation of vapor at lower (0.0, 0.16 and 0.40 mg/L) doses; inhalation of aerosol at highest dose (1mg/L)

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1.4 m³ stainless steel and glass NYU-style inhalation chamber
- System of generating particulates/aerosols: vapour generation: flash-evaporation in a tube maintained at 250-300°C; aerosol vapour: nebulizer
- Air flow rate:300 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: sample collection: in acetone-filled impingers; analysis method: GC-FID
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug day 1 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:

Samples of atmospheric DBE were taken from the rat breathing zone at approximately 60-minute intervals by drawing calibrated volumes of chamber atmosphere through fritted glass midget impingers containing acetone. Samples were analyzed using a Hewlett-Packard Model 5710 Gas Chromatograph (GC) equipped with a flame ionization detector. DBB was chromatographed isothermally at 150°C on a 3 ft x 2 mm ID glass column packed with 10% SP-1000 on Chromasorb W-AW 100/120 mesh.

Chamber concentrations were determined by comparing the chamber sample GC response with that obtained from standard samples prepared by quantitative dilution of DBE in acetone. The DBE chamber concentration was calculated after measuring the peak area of the largest DBE component, dimethyl glutarate. In addition, the relative amounts of the 3 largest DBE components (dimethyl-glutarate, -adipate, and -succinate) were qualitatively compared for each sample.

Duration of treatment / exposure:

pre-breeding: 6 h per day
breeding, gestation, lactation: 6 h per day;

Frequency of treatment:

pre-breeding: 5 days per week, 14 weeks;
breeding, gestation, lactation: 7 days per week, 8 weeks;

Remarks:

Doses / Concentrations:
0.0 (control), 0.16, 0.40, 1.0 mg/L
Basis:
nominal conc.

No. of animals per sex per dose:

20 males;
20 females;

Control animals:

yes, concurrent no treatment

Details on study design:

Groups of 20 male and 20 female rats were exposed to DBE at concentrations of 0 (control), 0.16, 0.40 (maximum attainable vapor), or 1.0 mg/L (aerosol) in whole-body inhalation chambers. Exposures were conducted for 6 hours/day, 5 days/week for 14 weeks (pre-breeding) then 7 days/week for 8 weeks (through breeding, gestation, and lactation). The exposures were interrupted for female rats between gestation day 19 and postpartum day 3. Parental examinations included clinical and cage side observations, body weight determination, and assessment of mating performance, fertility, gestation length, and lactation performance. The number and sex of pups, viability, and weight were determined. At the end of the lactation period, all parental rats and ten 21-day old pups of each sex were per group were killed and examined for gross abnormalities. Parental nose tissues were examined histopathologically.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

OTHER:
Mating performance, fertility, gestation length, and lactation performance were assessed.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, viability, weight

Postmortem examinations (parental animals):

All parental animals were sacrificed by exanguation under chloroform anesthesia and examined for gross anatomic abnormalities. Additionally, the nasal tissues were examined histologically; other tissues were examined grossly and saved. Mean organ and organ-to-body ratios were calculated for the brain, heart, lungs, liver, spleen, kidneys, testes and thymus.

Postmortem examinations (offspring):

Ten 21-day old arbitrarily selected pups of each sex per dose group were sacrificed by exanguination under chloroform anesthesia and examined for gross anatomic abnormalities. Mean organ and organ-to-body ratios were calculated for the brain, liver, kidneys, and testes.

Statistics:

For parental organ and body weight analyses, data were statistically analyzed by a one-way ANOVA. When the ratio of variance (F) indicated a significant group variation, test groups were compared with appropriate control group by least significant difference test for body weight data and by Dunnett's test for organ and final body weight data. Litter data were analyzed by Mann-Whitney U-test. Significance for statistical tests was judged at the 0.05 probability level.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

upper respiratory tract irritation

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHTS AND CLINICAL SIGNS
Body weights were decreased in the 0.40 mg/L group female rats during the last week of the study. Weights were slightly decreased in male and female rats in the 1.0 mg/L group starting around the seventh week of the study. No unusual signs or behaviours that could be associated with exposure to DBE were seen in rats in any of the treated groups. Parental rats in the 1.0 g/L group showed wet fur during exposures from aerosol deposition. The fur dried about 2 hours after exposure.

REPRODUCTION PARAMETERS
No treatment-related differences were observed between the control and test groups with regard to male or female fertility, gestation length, litter sizes, viability, or lactation performance. A slight but statistically significant decrease in viability index at birth (number of pups born alive per number of pups born) was observed in the 0.16 mg/L DBE group but this was considered to be unrelated to DBE exposure because it was not seen at either of the two higher exposure concentrations. All pups delivered by both DBE-exposed and control rats appeared outwardly normal. Body weights at birth and weaning (21 days pp) were significantly lower in the 1.0 mg/L group. The fertility rate in the control group was lower than that of the test groups (60% versus 75 to 90% for the test groups) and is low compared to historical rates for this laboratory. One potential contributing factor to the low fertility rate in the control group is that there were some males that never had the opportunity to mate with a subsequently proven fertile female; three of these males were in the control group, three in the 0.4 mg/L group, and two were in the 0.16 mg/L group. On the other hand all the males in the 1 mg/L group were mated with females who were subsequently proven to be fertile. Although the cause is uncertain, the control group low fertiIity rate did not impact the toxicity evaluation since the fertility rate in the DBE-exposed groups was well within historical limits.

PATHOLOGY
No gross pathologic changes were seen in any of either the parental rats or their offspring. Histopathologic evaluation of parental generation rat nasal tissues showed squamous metaplasia primarily in the olfactory epithelium in all groups exposed to DBE. The nasal effect was minimal in the 0.16 mg/L group rats and of mild to moderate severity in the 0.40 and 1.0 mg/L groups. The squamous metaplasia was characterized by a flattening and pavementing of epithelial cells which replaced the normal architecture of olfactory epithelium. In some cases, particularly in the 0.40 and 1.0 mg/L rats, this squamous change was accompanied by a very minimal to mild suppurative inflammation. The squamous metaplasia was present primarily in the olfactory epithelium of the dorsal meatus, along the dorsal portion of the nasal septum, and on the tips of the ecto- and endoturbinates in the nasal cavity. There was also an increase in squamous metaplasia of the respiratory epithelium in the nasal cavity in the high-dose rats. The severity of the lesions ranged from absent-to-minimal up to moderate in some rats. One male rat in the 1.0 mg/L group had a meningeal sarcoma surrounding the olfactory region of the brain. Because the tumor did not communicate with the nasal cavity and the tumor cell type was unrelated to any nasal epithelial cell types, the tumor was considered to be unrelated to inhalation of DBE.

ORGAN WEIGHTS
In parental rats, liver-to-body weight ratios were slightly lower than the controls in the rats exposed to either 0.40 and 1.0 mg/L. Other incidental differences between test and control rats included slight decreases in absolute heart and kidney weights in female rats in the 0.40 and 1.0 mg/L groups. A slight decrease in absolute spleen weight and a slight increase in relative brain weight were observed in females in the 1.0 mg/L group. These differences were not dose-related and may have been related to the slight body weight differences between the test and control groups and were considered of minimal biological significance.

Key result

Dose descriptor:

NOEC

Remarks:

for reproductive parameters

Effect level:

> 1 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Key result

Dose descriptor:

LOAEC

Remarks:

for local toxicity effects

Effect level:

0.16 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the nasal histopathology data

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

A slight decrease in relative kidney weight was seen in female pups whose parents were exposed to 0.40 mg/L of DBE; however, in the absence of a dose-response relationship, the decreased relative kidney weights were not considered to he related to DBE exposure. No differences were seen in the liver weights of DBE-exposed pups.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

> 1 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Key result

Reproductive effects observed:

no

Reproduction parameters in rats exposed to DBE by inhalation

Parameter	DBE Concentration [mg/L]
	0 (control)	0.16	0.40	1.0
Male fertility [%]	12/20 60	16/20 80	15/20 75	18/20 90
Female fertility [%]	17/20 85	17/20 85	17/20 85	20/20 100
Viability index at birth	100	94.6 *	99.6	99.0
Viability index day 0-4	99.5	93.7	99.2	98.2
Lactation index	100	100	97	99
Gestation index	100	100	100	100
Pups / litter [mean]	13.1	12.5	12.6	12.9
Gestation duration [days]	22.1	22.1	22.1	21.8
Pup weight at birth [g]	6.4 (0.6)	6.4 (0.8)	6.1 (0.1)	5.9 (0.1) *
Pup weight at day 21 [g]            Male            Female	44.3 (2.6) 41.0 (3.9)	44.9 (4.3) 41.7 (4.7)	45.5 (4.1) 44.3 (4.2)	41.8 (2.8) * 40.8 (2.0) *

 * Significantly different to control, p < 0.05

Values in parentheses report standard deviation

Conclusions:

No effects on reproduction parameters were observed in rats exposed by inhalation up to 1.0 mg/L DBE, a concentration that produced both body weight and histologic effects in parental rats.

Executive summary:

Dibasic Esters (DBE) has been tested in a reproduction toxicity study on Crl:CD(SD)BR rats after inhalation exposure using a protocol similar to OECD guideline no 415 and EU method B34 in compliance with US TSCA Good Laboratory Practice.

Groups of 20 male and 20 female rats were exposed to DBE at concentrations of 0 (control), 0.16, 0.40 (maximum attainable vapor), or 1.0 mg/L (aerosol) in whole-body inhalation chambers. Exposures were conducted for 6 hours/day, 5 days/week for 14 weeks (pre-breeding) then 7 days/week for 8 weeks (through breeding, gestation, and lactation). The exposures were interrupted for female rats between gestation day 19 and postpartum day 3. Parental examinations included clinical and cage side observations, body weight determination, and assessment of mating performance, fertility, gestation length, and lactation performance. The number and sex of pups, viability, and weight were determined. At the end of the lactation period, all parental rats and ten 21-day old pups of each sex were per group were killed and examined for gross abnormalities. Parental nose tissues were examined histopathologically. No significant differences were observed between control and test rats with respect to mating performance, fertility, length of gestation, or progeny numbers, structure, and viability. Body weights of parental rats and of their offspring were reduced at 1.0 mg/L. The only histopathologic changes detected in the nasal tissues of the parental rats, was an exposure-related increase in squamous metaplasia in the olfactory epithelium. There was an increase in liver-to-body weight ratios in the two higher parental exposure groups and an increase in the lung-to-body weight ratio also seen at 1.0 mg/L. From the results obtained, it can be concluded that reproduction in rats was not altered by repeated inhalation exposure to up to 1.0 mg/L DBE, a concentration that produced both body weight and histologic effects in parental rats.

 

Dibasic Esters (DBE) is not classified as reproductive toxicant according to the criteria of Directive 67/548/EC and EU Regulation No. 1272/2008 (CLP).

Effect on fertility: via oral route

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on fertility: via inhalation route

Dose descriptor:

NOAEC

1 000 mg/m³

Additional information

There are no data available on the reproductive toxicity of the reaction mass of dibutyl esters of adipic and glutaric acid. However data exist for the methyl esters of adipic, glutaric and succinic acids, and for the dibutyl adipate. Dosing of the dibutyl esters will result in the release of the acids and butanol, therefore read-across to the dimethyl esters is considered apropriate since the major hydrolysis product of the dimethyl esters is the acids. Further information on the read-across justification is provided in the document in section 13 and section 0.

Data on the Dimethyl esters:

The effects of dibasic esters on fertility were assessed by the inhalation route in a one-generation study (equivalent to an OECD 415 study) in rats, exposed to to the test concentrations of 0, 160, 400 or 1000 mg/m3 for 14 weeks pre-bredding, and for 8 weeks through breeding, gestation and lactation. No relevant effects occurred in mating performance, fertility, gestation duration, litter size, development or viability, and lactation performance up to the highest concentration tested. The NOEC for reproductive toxicity was therefore 1000 mg/m3.

 

Furthermore, changes in sex hormones were observed in the repeat-dose inhalation toxicity study in rats exposed to each component of the dibasic ester blend for 90 days (for details, see "7.5.3. Repeated dose toxicity: inhalation" section). Increased epididymal sperm counts were noted in male rats exposed to 400 mg/m3 dimethyl adipate, 400 mg/m3 dimethyl succinate, and 50 or 400 mg/m3 dimethyl glutarate. A statistically significant decrease in serum estradiol concentrations was noted in female rats exposed to 400 mg/m3 dimethyl succinate. A significant decrease in serum testosterone concentrations was noted in male rats exposed to 50 or 400 mg/m3 dimethyl glutarate. Serum luteinizing hormone (LH) concentrations were also significantly decreased at 400 mg/m3 dimethyl glutarate. The toxicological significance of these changes was unclear, as a decrease in male sex hormones should have resulted in a reduction of epididymal sperm counts, and yet the opposite was observed. In addition, none of these effects were observed in the dedicated fertility study using the dibasic ester blend. These hormonal variations can therefore be considered incidental.

Data on Dibutyl adipate:

In addition to the data on the dimethyl esters there is a reproductive developmental screening study (OECD 421) on dibutyl adipate. In this study there were no observed effects at the highest dose tested (1000 mg/kg bw/day) on reproductive performance parameters including fertility, gestation length and number of implantations. In the top dose group the study authors indicate there was a small effect on pup survival between post natal day 1 and 4. This was also associated with a small effect on pup weights in this dose group between post natal days 1 and 4. However, in scrutinising the study data it is clear that these apparent effects are only just reaching statistical significance and appear to represent a statistical anomaly rather than a real toxicological effect. As such the NOEL for reproductive effects is actually considered to be 1000 mg/kg bw/day.

Short description of key information:

A one-generation reproductive toxicity study is available for the methyl esters of adipic, succinic and glutaric acids. An OECD 421 is available for dibutyl adipate.

Effects on developmental toxicity

Description of key information

Inhalation developmental toxicity studies are available for the reaction mass of methyl esters of adipic, glutaric and succinic acid, and dimethyl glutarate. In addition to these studies, data on the glutaric and adipic acids, and n-butanol are available to support the assessment.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Kingston, NY
- Age at study initiation: females: 63 days, males: 84 days
- Weight at study initiation: females: 146-198g; males: 303-396g
- Housing: individually in stainless steel wiremesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1.4 m3 stainless steel and glass NYU-style inhalation chamber
- Method of holding animals in test chamber: in cages
- System of generating particulates/aerosols: 0.16 and 0.4 mg/L: liquid DBE was metered into a furnace maintained at 50-300°C; for the 1.0 mg/L concentration, mixed aerosol/vapour athmospheres were generated using a Spraying Systems Nebulizer
- Temperature, humidity, pressure in air chamber:
- Air flow rate: about 300 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: GC-FID
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were analyzed using a Hewlett-Packard Model 5710 Gas Chromatograph (GC) equipped with a flame ionization detector. DBE was chromatographed isothermally at 150°C on a 3' x 2 mm ID glass column packed with 10% SP-1000 on Chromosorb W-AW 1001120 mesh.

Details on mating procedure:

The female rats were cohabited overnight with mature males (1:1). Mating was verified each morning by detection ol a copulation plug in the vagina or on the cage hoard. The day a plug was found was designated Day 1 of gestation (Day 1G). Mated females were stratified by body wcight and assigned to groups by random sampling within each stratum (24 per group).

Duration of treatment / exposure:

In the definitive study, groups of 24 pregnant rats were targeted for exposure to 0 (control), 0.16, 0.4, and 1.0 mg DBE /L. Rats were exposed to whole-body inhalation for 6 hr per day from Days 7 through 16 (10 exposures). The control dams were exposed to air only.

Frequency of treatment:

6h per day

Duration of test:

Day 7 to 16 (10 exposures)

Remarks:

Doses / Concentrations: 0.0, 0.16, 0.4, 1.0 mg/L
Basis: nominal conc.

Remarks:

Doses / Concentrations: 0.0, 0.15, 0.38, 0.99 mg/L
Basis: analytical conc.

No. of animals per sex per dose:

24 pregnant rats per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a pilot study

Maternal examinations:

Body weights and clinical signs were recorded on the day after arrival and before mating; observations for morbidity and mortality were made daily. Females selected for the study were weighed on Days 1, 7, 9, 11, 13, 15, 17, and 21G. Feed was weighed on Days 1, 3, 9, 11, 13, 15, 17, 19 and 21G and each afternoon on Days 7-16G (the exposure period). Just before euthanasia on Day 21G. each dam was coded, so personnel involved did not know the exposure group to which any dam or fetus belonged. Dams were euthanized by cervical dislocation, a gross pathologic examination was made, and the liver was weighed. The ovaries were removed and the number of corpora lutea were counted at a magnification of 2.5 x (Ednalite).

Ovaries and uterine content:

The uterus was opened and the number and position of all live, dead, and resorbed conceptuses were recorded. Both gravid and empty uteri were weighed. The uterus of each apparently nonpregnant dam was stained with ammonium sulfate solution to determine whether any very early resorption could be identified.

Fetal examinations:

All fetuses were weighed and examined for external alterations at a magnification of 2.5x. About one-half of the live fetuses in each litter (plus all stunted and malformed fetuses) were examined for visceral alterations. For each litter, the maximum stunted weight (MSW) was calculated by subtracting the lightest weight from the total weight dividing by the remaining number of fetuses, and multiplying by 0.666. A fetus weighing the same or less than the MSW was considered stunted, and its weight was omitted after the mean litter weight was calculated. The heads of these fetuses were fixed in Bouin’s solution and examined. All fetuses were sexed internally and then they were fixed in 70% ethanol, eviscerated, macerated in 1% aqueous KOH, and stained with alizarin red S to permit examination of the skeletons for alterations.

Statistics:

The litter was considered the experimental unit for the purpose of statistical evaluation. The level of significance selected was 0.05.

Details on maternal toxic effects:

Details on maternal toxic effects:
All female rats survived the testing period. The number of pregnant rats/group was not altered by exposure to DBE, with the numbers being 24, 21, 20, and 24, control to high concentration groups, respectively. Body weight changes in maternal rats exposed to either 0.4 or 1.0 mg/L, were reduced, whereas those from the 0.16 mg/L were not. Feed consumption was reduced in the 0.4 and 1.0 mg/L group rats during the first 6 days of exposures (means of 23.3, 20.5, and 20.1 g/day, 0, 0.4, and 1.0 mg/L groups, respectively). Clinical observations of perinasal staining (15 rats) and wet fur (20 rats) were seen in the 1.0 mg/L group; while these findings were not seen in the controls. Perinasal staining was seen in 1 rat from the 0.16 mg/L group and in 4 rats from the 0.4 mg/L group; wet fur was also seen in 1 rat from the 0.4 mg/L group. Other clinical observations including alopecia, sores, periocular and facial staining, and swollen extremities were seen but infrequently and with no suggestion of a relationship to DBE exposure. No significant differences in absolute or relative liver weight was found but a significant trend in absolute weight was evident; a trend not seen when related to the body weight where values for all test groups were lower (but not significantly) from that of the controls.

Key result

Dose descriptor:

LOAEC

Effect level:

0.16 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: perinasal staining in all dose groups

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No adverse reproductive effects related to DBE exposure were detected. There was no effect on fetal weights at any exposure level. There was no significant difference between the control and experimental groups in the incidence of external, visceral, or skeletal malformations. Developmental variations among fetuses derived from DBE-exposed female were not significantly different from those of the controls.

Key result

Dose descriptor:

NOEC

Effect level:

> 1 mg/L air

Based on:

test mat.

Sex:

female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

DBE exerts no adverse effect on prenatal development in the rat following inhalation exposures as high as 1.0 mg/L DBE during the period of organogenesis.

Executive summary:

Dibasic Esters (DBE) has been tested in a developmental toxicity study on Crl:CD(SD)BR rats after inhalation exposure using a protocol similar to OECD guideline no 414 in compliance with Good Laboratory Practice.

Pregnant Crl:CD BR rats (7-8 per group) were exposed to either 0.16, 0.4, or 1.0 mg/L DBE by inhalation for 6 hr/day from Days 7 through 16 of gestation (day in which copulation plug was detected was designated Day 1G) in whole-body inhalation chambers. A control group of pregnant rats was exposed simultaneously to air only. Parental examinations included clinical and cage side observations, body weight determination, and determination of feed consumption. Gross pathologic examination was performed on day 21G and included counting of the corpora lutea and determination of number and position of all live, dead, and resorbed conceptuses in the uterus. Liver, gravid and empty uteri were weighed. All fetuses were weighed and examined or external alterations. About one-half of the live fetuses were examined for visceral alterations. All fetuses were sexed and examined for alterations of the skeleton after maceration. A suppression of both food consumption and the rate of body weight gain was seen in the 0.4 and 1.0 mg/L groups during the first 6 exposure days. Staining on the fur and perineal area was seen in rats exposed to 1.0 mg/L and liver weight decreases, although not statistically significant, occurred in the 2 high exposure groups. None of the reproductive parameters were altered in any of the groups and no fetal effects were detected.

 

DBE exerts no adverse effect on prenatal development in the rat following inhalation exposures as high as 1.0 mg DBE during the period of organogenesis.

Based on the given data, dibasic esters (DBE) is not classified as reproductive toxicant according to the criteria of Directive 67/548/EC and EU Regulation No. 1272/2008 (CLP).

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Study period:

July 3, 2002 - April 15, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

according to EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: Hra:(NZW)SPF

Details on test animals or test system and environmental conditions:

The rabbit (Hra:(NZW)SPF) was selected for this study to provide data in a non-rodent species and because it is a preferred species for developmental toxicity testing. The Hra:(NZW)SPF strain was chosen because extensive background information is available from the literature, the supplier, and previous studies with other compounds at Haskell Laboratory. A total of 110 nulliparous, time-mated female rabbits were received from COVANCE, Denver, Pennsylvania. Sixty-six female rabbits were received on June 19, 2002 and 44 female rabbits were received on June 21, 2002. Body weights on the day of mating defined as day 0 of gestation (G) were supplied by the vendor. The rabbits for this study were approximately 6 months old and were at either day 1, 2, or 3G upon arrival..

Animal rooms were maintained at an acceptable temperature of 17-23°C (targeted at 20°C) and a targeted relative humidity of 50% ± 10%. Animal rooms were artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours). Animals were housed individually in suspended, wire-mesh, stainless steel cages. Nesting material was not provided because the does were euthanized prior to parturition. To address logistical considerations, the rabbits on this study were housed in 4 animals rooms near the exposure chambers. There was a significant aerosol component in the high level
chamber; therefore, to prevent unintentional exposure of control or lower level animals to the residual aerosol, the control group rabbits were housed separately in one room. The rabbits exposed to 1000 mg/m³ were housed separately within a hood so that the higher air flow in the hood promoted evaporation of aerosol deposits on the fur. The remaining 3 groups of rabbits were housed in 2 other rooms.

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Spraying Systems Nebulizer
- Method of holding animals in test chamber: individually restrained in wire mesh cages
- Source and rate of air: house supply
- Method of conditioning air: Cole Palmer Masterflex® Console Drive pump
- System of generating particulates/aerosols: Brooks model 5851i Mass Flow Controller
- Temperature, humidity, pressure in air chamber: 17-23°C at 50 ± 10% humidity
- Air flow rate:
- Air change rate:
- Method of particle size determination: Sierra® Series 210 Cyclone Preseparator/Cascade Impactor and Sierra® Series 110 Constant Flow Air Sampler
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: 0, 30 100, 300, 1000 mg/m3
- Lot/batch no. of vehicle (if required):
- Purity of vehicle: NA

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For all test exposure chambers, the vapor component of the atmospheric concentration of DMG was determined by gas chromatography. For the test exposure chambers, known volumes of chamber atmosphere were drawn, at approximately 90-minute intervals, from the breathing zone of the animals through glass impingers containing acetone as the collection medium. The control chamber was similarly sampled once daily for measurement of atmospheric concentration. Aliquots of impinger solution were injected on a Restek® fused silica glass (inside diameter 0.53 mm) with RTX-502.2 coating column in a Hewlett-Packard model 6890 Series Gas Chromatograph equipped with a flame ionization detector. The atmospheric concentration of the test substance was determined from a standard curve derived from liquid standards. Standards were prepared weekly by the quantitative dilution of DMG liquid in known volumes of acetone. For the 300 and 1000 mg/m³ exposure chambers, the aerosol component of the atmospheric concentration of DMG was determined by gravimetric analysis. Known volumes of chamber atmosphere were drawn, at approximately 30-minute intervals, from the breathing zone of the animals through a 25 mm filter cassette that contained a pre-weighed Gelman glass fiber (Type A/E) filter. The filters were weighed on a Cahn model C-31 Microbalance®. The filter weights were automatically transferred to CITADS, which calculated the chamber concentrations based on the difference in pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled. Gravimetric sample start- and stop-times for each sample were also controlled and recorded by CITADS. Prior to study start, gravimetric samples collected from the 30 and 100 mg/m³ chambers indicated that no aerosol was present for these 2 concentrations. A sample to determine particle size distribution (mass median aerodynamic diameter and percent particles less than 1, 3, and 10 µm diameter) was taken once per week from the 1000 mg/m³ exposure chamber with a Sierra® Series 210 Cyclone Preseparator/Cascade Impactor and Sierra® Series 110 Constant Flow Air Sampler.

2. Environmental Monitoring
Chamber airflow

Details on mating procedure:

Mating details not provided. A total of 110 nulliparous, time-mated female rabbits were received from COVANCE. Sixty-six female rabbits were received on June 19, 2002 and 44 female rabbits were received on June 21, 2002.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

Daily during days 7-28G

Duration of test:

22 days

Remarks:

Doses / Concentrations:
0, 30, 100, 300, 1000 mg/m3 (for the 1000 mg/m3 group the mean daily vapor concentrations ranged from 450-590 mg/m³, and the mean daily aerosol concentrations ranged from 410-580 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

22

Control animals:

yes

Maternal examinations:

1. In-life Observations of Females
a. Body Weight
Body weights were recorded in the morning on the day after arrival through day 29G.
b. Food Consumption
Food consumption was measured daily on days 4-29G.
c. Clinical Signs
Clinical signs were recorded each morning on days 4-29G and each afternoon following
exposures on days 7-28G. Observations, at approximately hourly intervals, were made during
the exposures to assess, to the extent possible, the overall state of the animals. In addition, the
response of the animals to a sound stimulus while in the chambers was recorded before vapor
generation began, after approximately 3 hours of exposure, and prior to removal of the animals
from the chambers.

2. Postmortem Observations of Females Dying Prior to Scheduled Euthanasia
A gross external and visceral examination was performed within 24 hours after the doe was
found dead or sacrificed in extremis. Pregnancy status was determined and the uterine contents
were described. Dead rabbits were refrigerated until necropsied.

3. Postmortem Observations of Does Aborting or Delivering Early
a. Dam Observations
Dams that aborted (day 27G or earlier) or delivered early (day 28 or 29G) were euthanized within
24 hours of detection and a gross external and visceral examination was performed.
b. Fetal Observations
Fetuses remaining in the uterus or aborted/delivered early were examined to the extent possible.
Data from these litters were maintained with the raw data but were excluded from all relevant
fetal data calculations.

4. Postmortem Observations of Does Surviving to Scheduled Euthanasia
a. External Appearance
Each doe was examined immediately after euthanasia on day 29G.
b. Viscera
Viscera were examined grossly immediately after euthanasia.
c. Uterine Weight
The intact and empty uterus of each doe having at least one viable fetus was weighed to permit
calculation of maternal body weight adjusted to exclude the products of conception.
d. Corpora Lutea
The count for each ovary of dams with viable fetuses was recorded.
e. Implantations
For each female with visible implants, the type (live and dead fetuses, and resorptions) and their
relative positions were recorded. The uterus of each apparently "nonpregnant" female was
stained with ammonium sulfide to detect very early resorptions.

Ovaries and uterine content:

see materal examinations

Fetal examinations:

Fetuses of Does Surviving to Scheduled Euthanasia
a. Number, Location, and Condition
These parameters were recorded for each fetus.
b. Fetal Weight
The body weight for each fetus was recorded.
c. External Alterations
The alterations detected for each live fetus was recorded.
d. Soft Tissue Alterations
For each live fetus, fetal sex and any visceral alterations detected were recorded.(5) A transverse
section, deep enough to permit examination of the brain, was made between the parietal and
frontal bones. On the day of sacrifice, the eyelids of each fetus were removed to examine the
eyes for alterations.
e. Skeletal Alterations
After fixation in alcohol, the alizarin-stained skeletons were examined and alterations detected
were recorded for each live fetus.

Statistics:

Statistical Analyses
Descriptive statistics including mean, standard deviation, and standard error of the mean were
used to summarize exposure concentration and environmental data.
For the parameters listed below, descriptive statistics were performed and sequential trend tests
were applied to the data as tabulated below.(6) If a significant dose-response trend was detected,
data from the top dose group were excluded and the test repeated until no significant trend was
evident. For litter parameters, the proportion of affected fetuses per litter or the litter mean was
used as the experimental unit for statistical evaluation.(7) The level of significance selected was
p < 0.05.
Parameter Trend Test
Maternal weight Linear contrast of means(8)
Maternal weight changes
Maternal food consumption
Live fetuses Jonckheere's test(9)
Dead fetuses
Resorptions
Nidations
Corpora lutea
Incidence of fetal alterations
Incidence of pregnancy Cochran-Armitage test(8)
Clinical observations
Maternal mortality
Females with total resorptions
Abortions/early deliveries
Fetal weight Linear contrast of least square means(10)
Sex ratio
Where the data were tied and the standard large sample version of Jonckheere's test was not
applicable, exact p values for Jonckheere's test were calculated using permutation
methodology.

Indices:

Maternal weight
Maternal weight changes
Maternal food consumption
Live fetuses
Dead fetuses
Resorptions
Nidations
Corpora lutea
Incidence of fetal alterations
Incidence of pregnancy
Clinical observations
Maternal mortality
Females with total resorptions
Abortions/early deliveries
Fetal weight
Sex ratio

Details on maternal toxic effects:

Details on maternal toxic effects:
1. Mortality and Reproductive Outcome
There was no compound-related mortality at exposure levels ≤ 300 mg/m3. At 1000 mg/m3, one female was found dead on day 13G and another female was sacrificed in extremis on day 22G. There were no compound-related effects on indicators of reproductive outcome. One control group rabbit aborted on day 19G and one 100 mg/m3 group rabbit delivered early prior to euthanasia on day 29G. In addition, one 1000 mg/m3 litter consisted entirely of late resorptions.
The findings for this one litter were not considered to be compound-related because no other litters were similarly affected and additionally, there was no increase in resorptions for litters also containing live fetuses. At 1000 mg/m3, there was a significant decrease in the mean number of nidation sites per doe. This was not considered related to the exposure because implantation is considered to have occurred prior to the start of exposures on day 7G. Furthermore, this decrease is consistent with a lower mean corpora lutea count in this group.

2. Body Weights and Weight Changes
There were no compound-related effects on maternal body weight parameters at exposure levels ≤ 100 mg/m3. There were significant reductions in overall weight change (days 7-29G) at 300 and 1000 mg/m3; these significant effects were evident at the onset of exposures (days 7-9G) and generally persisted throughout gestation.

3. Food Consumption
There were no compound-related effects on maternal food consumption at exposure levels ≤ 300 mg/m3. At 1000 mg/m3, mean food consumption was significantly reduced when averaged over the exposure period (days 7-29G); the reduction was significant and pronounced at the onset of exposures (days 7-9G) and persisted throughout exposures to some extent as well.

4. Clinical Observations
There were no compound-related increased clinical observations at exposure levels ≤ 100 mg/m3. At 300 and 1000 mg/m3, compound-related clinical observations included clear ocular discharge; at 1000 mg/m3, all animals had wet fur.

5. Postmortem Findings
There were no compound-related gross postmortem findings at any level tested.

Key result

Dose descriptor:

NOAEL

Remarks:

100 mg/m3

Effect level:

> 0 - <= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:

Fetal Findings
1. Mortality and Sex Ratio
There were no compound-related effects on embryofetal viability or on fetal sex ratio at any level tested. The mean number of live fetuses per litter appeared to be slightly reduced at 1000 mg/m3, however, this apparent reduction was not considered compound-related and is believed to have resulted from previously discussed reductions in mean numbers of corpora lutea and nidations at this level that were not considered compound-related.

2. Body Weight
There was no compound-related effect on fetal body weight at any level tested.

3. Malformations
There were no compound-related fetal malformations at any level tested.

4. Variations
There were no compound-related fetal variations at any level tested. There was a significant increase in retarded sternebral ossification at 1000 mg/m3. This increase, however, was not considered to be compound-related; rather, the statistical significance appears to be the result of an unusually low control group value relative to relevant historical control group data.

Key result

Dose descriptor:

NOAEC

Effect level:

> 1 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

female

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the conditions of the current study, there was no evidence of compound-related maternal
toxicity at 30 or 100 mg/m3 and there was no evidence of developmental toxicity at any level
tested. Thus, the no-observed-effect level (NOEL) a for maternal toxicity was considered
100 mg/m3 and the NOEL for developmental toxicity was considered 1000 mg/m3.

Executive summary:

Dimethyl glutarate (DMG) was administered by inhalation to groups of 22 time-mated Hra:(NZW)SPF rabbits. The inhalation exposures were whole-body exposures and animals were exposed for approximately 6 hours each day on days 7 through 28 of gestation. Target chamber concentrations were 0, 30, 100, 300, or 1000 mg/m3. During the in-life period, maternal body weight, food consumption, and clinical observation data were collected daily. Animals were euthanized on day 29 of gestation; dams were subjected to a gross external and internal postmortem examination. Gravid uteri were removed, weighed, and dissected and the uterine contents were described. Each live fetus was weighed, sexed, and examined for external, visceral, and skeletal alterations. At 1000 mg/m3, there was compound-related mortality; one animal was found dead on gestation day 13 and one animal was sacrificed in extremis on gestation day 22. Among survivors, there were compound-related reductions in maternal body weight gains and food consumption and there were compound-related clinical observations (ocular discharge and wet fur). At 300 mg/m3, evidence of compound-related maternal toxicity was limited to reduced maternal body weight gains and increased clinical observations (ocular discharge). Under the conditions of the current study, there was no evidence of compound-related maternal toxicity at 30 or 100 mg/m3 and there was no evidence of developmental toxicity at any level tested. Thus, the no-observed-effect level (NOEL)a for maternal toxicity was considered 100 mg/m3 and the NOEL for developmental toxicity was considered 1000 mg/m3.

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

GD 6-15

Frequency of treatment:

daily

Duration of test:

GD6-20

Remarks:

Doses / Concentrations:
0, 2.9, 13, 62, 288 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Female/25, 25, 25, 25, 24 in the 0, 2.9, 13, 62, and 288 mg/kg groups, respectively

Control animals:

yes, concurrent no treatment

Dose descriptor:

NOAEL

Effect level:

> 288 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

> 288 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

The administration of up to 288 mg/kg of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. A summary of other reproductive outcomes

(represented as means/litter, except for resorptions and live litters) are provided in the table below:

Dose (mg/kg)	0	2.9	13	62	288
Copora Lutea	117	12.6	12.1	11.2	11.4
Implantations	11.4	11.3	10.6	11.1	11.5
Total No. Resorptions	2	6	3	0	7
Total No. Fetuses	11.2	11.0	10.3	11.1	11.2
Total No. live litters	20	23	24	22	20
Mean Fetal weight	3.88	3.89	3.83	4.01	3.99

The maternal and developmental NOAEL for this study was > 288 mg/kg.

Reference 4

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: appears to be well conducted, pre-dates guideline

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

Dutch

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

On Day 0, each doe was given an injection of human chorionic gonadotropin, and was artificially inseminated 3 hours later.

Duration of treatment / exposure:

Days 6-18 of Gestation

Frequency of treatment:

daily

Duration of test:

Days 6-18 of Gestation; Cesarean section Day 29

Remarks:

Doses / Concentrations:
0, 2.5, 12, 54, 250 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Female/19, 13, 16, 15, 20 in the 0, 2.5, 12, 54, and
250 mg/kg groups, respectively

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

The administration of up to 250 mg/kg of the test material to pregnant rabbits for 13 consecutive days had no clearly discernible effect on nidation, or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. A summary of other reproductive outcomes (represented as means/litter, except for resorptions and live litters) are provided in the table below:

Dose (mg/kg): 0 / 2.5 / 12 / 54 / 250

Corpora Lutea: 9.45 / 10.8 / 9.82 / 11.9 / 10.1

Implantations: 7.00 / 9.00 / 8.60 / 8.80 / 7.29

Total No. of Resorptions: 10 / 9 / 14 / 13 / 16

Total No. of Fetuses: 6.09 / 7.30 / 6.70 / 6.50 / 5.57

Total No. of Live Litters: 11 / 9 / 9 / 8 / 12

Mean Fetal Weight (g): 42.3 / 38.1 / 40.0 / 39.4 / 41.4

The maternal and developmental NOAEL for this study was > 250 mg/kg.

Conclusions:

adipic acid was not developmentally toxic in this assay

Reference 5

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

The rabbits used in the study were sexually mature stockbreeder males and sexually mature virgin females. The females were approximately 4-6 month of age, and their body weights ranged from 3.0-5.1 kg on gestation day 0. Each female was placed in a mating cage with a male (usually once in the morning and again in the afternoon) and after observation of copulation, vaginal smears were made and examined for the presence of motile spermatozoa (gestation day 0).

Duration of treatment / exposure:

Gestation Days 6-18

Frequency of treatment:

daily

Duration of test:

Gestation Days 6-18, Cesarean section Day 29

Remarks:

Doses / Concentrations:
0, 50, 160, 500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

18 per group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

No test substance-related mortality was observed. No changes in body weights or clinical signs were observed in females at any level tested. No adverse effects on pregnancy, and no embryotoxic or teratogenic effects were observed. A summary of reproductive outcomes is provided in the table below. All parameters, except pregnancy rate and sex ratio, are reported as means/litter. Concentration (mg/kg): 0 50 160 500

Pregnancy Rate (%): 94 94 83 83

Corpora lutea: 10 11 9 10

Implantations: 8 9 7 8

No. of Resorptions: 1.8 0.5 0.8 0.6

Total No. of Fetuses: 6 9 6 8

Mean Fetal Weight (g): 40.5 39.4 41.3 41.5

Sex Ratio (% male/female): 44/56 50/50 50/50 45/55

The maternal and developmental NOAEL for this study was > 500 mg/kg.

Conclusions:

No developmental toxicity observed in this study.

Reference 6

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

CD-1

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Male rats were 125 days old and mature virgin females were approximately 71 days old. Body weights of the female rats ranged from 190-262 g on Day 0 of pregnancy. Each male was housed nightly with up to 3 females until mating was completed. Vaginal washings were made on the morning after each exposure to a male, and the day of positive identification of spermatozoa in the washing was designated as day 0 of pregnancy (gestation day 0).

Duration of treatment / exposure:

Gestation Days 6-15

Frequency of treatment:

Daily

Duration of test:

Gestation Days 6-15, Cesarean section Day 20

Remarks:

Doses / Concentrations:
0, 125, 400, 1300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Females/25 per group

Control animals:

yes

Dose descriptor:

NOAEL

Effect level:

ca. 125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 1 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

No adverse effects were observed on body weight, general appearance, or behavior of rats at 125 mg/kg. At 400 mg/kg, no effect on body weight was observed, but salivation, rales, and nasal discharge were observed. At 1300 mg/kg, 1 death occurred (gestation day 10) and 1 animal was sacrificed early (gestation day 13). Mean body weight gains were decreased at 1300 mg/kg during the dosing period. The mean body weight gains post-dosing (gestation days 15-20) were normal compared to control, indicating a reversible effect on body weight after test substance withdrawal. Clinical signs observed at 1300 mg/kg included salivation, rales, nasal discharge, slight inactivity, labored breathing, decreased body temperature, soft stools, and staining around the mouth, nares, and anogenital area. No adverse effects on pregnancy, and no teratogenic effects were observed at any level tested. A significant increase in the number of resorptions at 1300 mg/kg was observed compared to control. The number or resorptions was within normal expected limits; therefore, the increase was not considered biologically meaningful. A summary of reproductive outcomes is provided in the table below. All parameters, except pregnancy rate and sex ratio, are reported as means/litter.

Concentration (mg/kg): 0 125 400 1300

Pregnancy Rate (%): 72 80 84 88

Corpora lutea: 15 15 15 15

Implantations: 13 14 14 13

No. of Resorptions: 0.4 0.9 0.5 1.0

Total No. of Fetuses: 13 13 13 12

Mean Fetal Weight (g): 3.6 3.7 3.7 3.6

Sex Ratio (% male/female): 52/48 48/52 51/49 52/48

The maternal NOAEL and LOAEL for this study were 125 and 400 mg/kg, respectively. The developmental NOAEL for this study was 1300 mg/kg.

Conclusions:

Glutaric acid was not developmentally toxic in this study

Reference 7

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare, Japan; Guidelines for Toxicity Studies of Drugs

Principles of method if other than guideline:

Pregnant rats were given drinking water containing 1-butanol at 0.2%, 1.0% or 5.0% (316, 1454 or 5654 mg/kg/day) on days 0–20 of pregnancy. The rats were sacrificed on day 20 of pregnancy and both the dams and fetuses were examined.

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tsukuba Breeding Center
- Age at study initiation: males: 10 weeks; females: 9 weeks
- Weight at study initiation: females: 217-273 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1-butanol was mixed with water to the according target concentrations. The stability of formulations in a dark and cool place under airtight conditions has been confirmed for up to 3 days. During use, the formulations were maintained under such conditions for no more than 3 days and were 95.7–103.5% of the target concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations were determined analytically and were 95.7–103.5% of the target concentration.

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: over night
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 0 through day 20 of pregnancy

Frequency of treatment:

continuous through drinking water

Duration of test:

until day 20 of pregnancy

Remarks:

Doses / Concentrations:
0.2%, 1.0% or 5.0% (316, 1454 or 5654 mg/kg/day)
Basis:
nominal in water

No. of animals per sex per dose:

20 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were determined based on the results of our range-finding study in which administration of 1-butanol in the drinking water on days 0–20 of pregnancy caused decreases in maternal body weight gain and food and water consumption and tended to reduce in fetal weight at 4% and 7% in rats.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not not specified


BODY WEIGHT: Yes
- Time schedule for examinations: once daily


FOOD CONSUMPTION: Yes
- Time schedule for examinations: every 3 or 4 days


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 10
- Organs examined: not specified

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- 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]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

The statistical analysis of fetuses was carried out using the litter as the experimental unit. The initial body weight, body weight gain and food and water consumption of pregnant rats, numbers of corpora lutea, implantations and live fetuses per litter, fetal weight and crown-rump length and placental weight were analyzed with Bartlett's test for homogeneity of variance at the 5% level of significance.
If it was homogeneous, the data were analyzed using Dunnett's multiple comparison test to compare the mean of the control group with that of each dosage group, and if it was not homogeneous, the mean rank of the 1-butanol-treated groups was compared with that of the control group with the Dunnett type test. The Dunnett type test was used for the incidences of pre- and postimplantation embryonic loss and fetal anomalies and sex ratio of fetuses to compare the mean rank of groups treated with 1-butanol and that of the control group. The incidence of dams with anomalous fetuses was analyzed by Chi-square test or Fisher's exact test. The significance of differences from the control group was estimated at probability levels of 1% and 5%.

Indices:

no data

Historical control data:

International Genetic Standard (Crj: CD (SD) IGS) rats were used throughout this study. This strain was chosen because it is most commonly used in reproductive and developmental toxicity studies and historical control data are available.

Details on maternal toxic effects:

Details on maternal toxic effects:
No death was found in female rats of any group. All females in all groups became pregnant. The body weight gains on days 0–7 of pregnancy were significantly reduced at 5.0%. The body weight gain during the whole period of pregnancy was also significantly decreased at 5.0%. No significant decrease in the body weight gain was noted at 0.2 or 1.0, except for a transient decrease on days 0–2 of pregnancy at 1.0%. The food consumption on days 0–7, days 7–14, days 14–20 and days 0–20 of pregnancy was significantly lower in the 1.0% and 5.0% groups than the control group. The water consumption on days 0–7 at 1.0 and 5.0% and on days 7–14, days 14–20 and days 0–20 at 5.0% was significantly decreased. The mean daily intakes of 1-butanol were 316 mg/kg for the 0.2% group, 1454 mg/kg for the 1.0% group and 5654 mg/kg for the 5.0% group.

Dose descriptor:

NOAEL

Effect level:

1 454 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No litters totally resorbed were found in any group. No effects of the administration of 1-butanol were observed on the numbers of corpora lutea, implantations, pre- or postimplantation loss, resorptions or dead or live fetuses or sex ratio of live fetuses. The body weights of male and female fetuses were significantly lower in the 5.0% group than in the control group. There was no significant difference in the crown-rump length of male and female fetuses or placental weight between the control and groups treated with 1-butanol.
One fetus with spina bifida in the control group and one fetus with thread-like tail and anal atresia in the 0.2% group were observed. Skeletal examination revealed one fetus with supernumerary thoracic vertebral bodies and malpositioned thoracic vertebrae at 1.0%. Although the total number of fetuses with skeletal variations was significantly increased at 5.0%, the number of fetuses with individual skeletal variations was not significantly increased, except for fetuses with short supernumerary ribs at 5.0%. A significantly lower number of forepaw proximal phalanges was observed at 5.0%. Membranous ventricular septum defect occurred in one fetus of the control and 0.2% groups and 3 fetuses in 3 dams of the 5.0% group. One fetus with a double aorta in the control group and one fetus with a left umbilical artery in the control and 2.0% groups were observed. Thymic remnants in the neck were found in 4–11 fetuses of the control and groups treated with 1-butanol.
However, there was no significant difference in the incidence of fetuses with internal abnormalities between the control and groups treated with 1-butanol.

Dose descriptor:

NOAEL

Effect level:

5 654 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

1 454 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Summary:

A significant decrease in maternal body weight gain accompanied by reduced food and water consumption was found at 5.0%. No significant increase in the incidence of pre- and postimplantation embryonic loss was observed in any groups treated with 1-butanol. Fetal weight was significantly lowered at 5.0%. Although a significant increase in the incidence of fetuses with skeletal variations and decreased degree of ossification was found at 5.0%, no increase in the incidence of fetuses with external, skeletal and internal abnormalities was detected in any groups treated with 1-butanol. The data demonstrate that 1-butanol is developmental toxic only at maternal toxic doses. No evidence for teratogenicity of 1-butanol was noted in rats. Based on the significant decreases in maternal body weight gain and fetal weight, it is concluded that the no observed adverse effect levels (NOAELs) of 1-butanol for both dams and fetuses are 1.0% (1454 mg/kg/day) in rats.

Reference 8

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Principles of method if other than guideline:

In a behavioural teratology study, exposed (6 wk) male rats were mated to non-exposed females. In another trial, non-exposed males and females were mated and subsequently the females were exposed. Functional and neurochemical observations were performed with the offspring (up to 90 d postnatal).

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

inhalation

Details on exposure:

Groups of 18 male Sprague-Dawley rats were exposed to concentrations of 0, 3000, or 6000 ppm (0, 9.2 or 18.5 mg/L) nBA for 7 hours/day for 6 weeks. These males were then mated to non-exposed female rats of the same strain. In a separate experiment, groups of 15 pregnant female rats were exposed to concentrations of 0, 3000, or 6000 ppm for 7 hours/day from gestation Day 1-20. These females were then allowed to deliver.

Duration of treatment / exposure:

day 1 - 20 of gestation

Frequency of treatment:

7 h/d

Remarks:

Doses / Concentrations:
0, 9.2 or 18.5 mg/L (= 0, 3000, 6000 ppm)
Basis:

No. of animals per sex per dose:

18 males/ 15 females

Control animals:

yes

Details on study design:

Sex: male/female
Duration of test: 20 days

Fetal examinations:

The offspring from these two groups were then observed for signs of developmental neurotoxic effects. Offspring were examined from postnatal days 10-90 for the following measures: ascent on a wire mesh screen, rotorod, open-field and photoelectrically-monitored activity, running wheel, avoidance conditioning and operant conditioning. Acetylcholine, dopamine, norepinephrine, serotonin, met-enkephalin, beta-endorphin, and Substance P. neurotransmitter levels were measured from the cerebrum, cerebellum, brainstem, and midbrain.

Dose descriptor:

NOAEL

Effect level:

18.5 mg/L air

Basis for effect level:

other: other:

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 9

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Principles of method if other than guideline:

Groups of approximately 15 Sprague-Dawley rats were exposed at 8000, 6000, 3500, or 0 ppm 1-butanol, for 7 hr/day on Gestation Days 1- 19 (sperm = 0). The highest concentration was selected to produce maternal toxicity. Dams were sacrificed on Gestation Day 20, and fetuses were individually weighed, tagged, and examined for external malformations. One-half of the fetuses were stained and examined for skeletal abnormalities, and the other half were examined for visceral defects using the Wilson technique.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Weight at study initiation: 176-200 g
- Housing: individually
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: 1-2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +- 2 °C
- Humidity (%): 50 +- 10 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation exposures were conducted in 0.5-m3 Hinners-type exposure chambers
- Method of holding animals in test chamber: Females were placed into 13 x 25 x 18-cm compartments in stainless-steel wire-mesh caging within the exposure chambers.

The vapor generation equipment was housed above the exposure chambers in glove boxes which were maintained under negative pressure to prevent any leakage of contaminants into the room. The test substance was placed into a flask. A low-flow pump circulated liquid from the reservoir flask into a 10-ml syringe contained within the flask such that the syringe was constantly overflowing.
Thus the syringe provided a constant head of chemical for a second pump (controlled by a micrometer adjustment) which injected the specified amount of liquid into a three-way valve which was attached to a Greensmith impinger. Heated compressed air was introduced through the second inlet of the three-way valve. Alcohol evaporation was controlled by regulating the preheating of compressed air. The impinger provided increased contact time between the air and the liquid to ensure total evaporation. In generation of high concentrations, glass beads were also placed at the bottom of the impinger to further increase the heat transfer area between the alcohol and the compressed air. This vapor and air mixture was introduced into the chamber airflow upstream of the orifice plate. The turbulence and pressure drop created by the orifice plate provided uniform mixing downstream of the vapor and air before the mixture entered the chamber. Airflow through the chambers provided approximately one air change per minute.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration within the chamber was monitored continuously with a Miran 1 A general-purpose infrared analyzer which was calibrated within the range to be tested.
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration within the chamber was monitored continuously with a Miran 1 A general-purpose infrared analyzer which was calibrated within the range to be tested.

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: until vaginal plug or sperim in vaginal smear was detected
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug and/or sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

day 1 - 19 of gestation

Frequency of treatment:

7 h/d

Duration of test:

until day 20 of gestation

Remarks:

Doses / Concentrations:
0, 10.8, 18.5 and 24.7 mg/l (0, 3500, 6000, 8000 ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

15-18 females per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were selected based on the results of an initial pilot study. For the teratology phase, the high concentration was selected to be maternally toxic, but not lethal, and two lower concentrations were included.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified
- Cage side observations: not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not specified


BODY WEIGHT: Yes
- Time schedule for examinations: Maternal weights were measured on Gestation Days 0, 7, 14, and 20. Females were also weighed each morning for the first week of exposure.


FOOD CONSUMPTION: Yes
Weekly food intake was measured on Gestation Days 0, 7, 14, and 20.


WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured on Gestation Days 0, 7, 14, and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with ovaries attached, no futher organs specified

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- 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]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

For the maternal data, multivariate analysis (with baseline as covariate) was used for weight comparisons across groups. The group differences in food and water intake were analyzed by multivariate analysis of variance. A Kruskal-Wallis test was used for group compansons of corpora lutea per animal. For the fetal data, analysis of variance was used to compare fetal weights across groups and sex. Group comparisons of the variables including litter size, percentage alive/litter, percentage normal/litter, and percentage females/litter were made using the Kruskal-Wallis test. For the variables including skeletal malformations, skeletal variations, visceral malformations, visceral variations, external malformations, and normormal fetuses, the number of litters with one or more of the variables of interest was compared between groups using Fisher's exact test. The results of the tests were adjusted for multiple comparisons, when appropriate, using the Bonferroni technique. A probability of p <= 0.05 was required for significance.

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Details on maternal toxic effects:
8000 ppm produced narcosis in approximately one-half of the dams. No behavioural effects were noted at 6000 ppm. Two of eighteen dams at 8000 ppm died during the exposure period. Feed consumption was decreased in the 6000 and 8000 ppm exposed dams (the statistical significance in the 8000 ppm group disappeared when adjusted for multiple comparisons), the 3500 ppm dams were similar to controls.
For 1-butanol, food consumption was reduced both at 6000 and 8000 ppm (overall means were 382 g for controls versus 320 and 332 g respectively). Water intake increased as pregnancy progressed and was generally higher, though not significantly, in treatment groups than in controls.

Dose descriptor:

NOAEL

Effect level:

10.8 mg/L air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No effect was observed on mean corpora lutea/litter, mean resorptions/litter, mean number of live foetuses/litter or sex ratio. Foetal weights were slightly decreased at 6000 and 8000 ppm groups, but the 3500 ppm group was unaffected. External foetal malformations were not observed. There were no differences in malformation rates (skeletal or visceral) or in rates of commonly observed variations. However, there was a slight increase in the percent of fetuses with any skeletal variation or malformation (mainly rudimentary cervical ribs) in the 8000 ppm group but not in the lower two exposure groups.

Dose descriptor:

NOAEL

Effect level:

24.7 mg/L air

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

10.8 mg/L air

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 10

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

See attached document with the justification for the category/read-across approach.

Principles of method if other than guideline:

Female rats were given aqueous solutions of n-butanol containing 0.24, 0.8 and 4% n-butanol (0.3; 1.0 and 5.0 g/kg/day) for 8 weeks before and during gestation. The control animals received tap water. The experiment was performed in two stages. The first comprised of the assessment of the oestrous cycle before exposure and then during 4-5 and 7-8 weeks of exposure, and the second stage of the fertility of female rats and their foetal development.

GLP compliance:

not specified

Species:

rat

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Own breeding colony (Imp:DAK)
- Age at study initiation:10 wks (females)
- Weight at study initiation: Females: 180-200 g
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22 °C
- Humidity (%): 45-55 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
the test substance was mixed with drinking water, the stability of the aqueous n-butyl alcohol solutions was assessed on several consecutive days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the aqueous n-butyl alcohol solutions was assessed on several consecutive days after their preparation using a Varian Aerograph 2800 gas chromatograph equipped with FID. The column was glass (2 in x 2 mm id .) packed with Porapak Q. The operating conditions were; carrier gas flow (nitrogen) 30 cm3/min ; hydrogen 30 cm3/min ; air 300 cm3/min; the temperature of the column, injection part and detector were 200°C, 200°C, 230°C, respectively. It was determined that the aqueous n-butyl alcohol solutions were stable within the concentration range used in the experiment (0.24-4%).

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: 3 weeks with untreated males
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

Exposure period: 8 weeks premating, mating (max. 3 weeks) , gestation day 0 - 20

Frequency of treatment:

daily, continuous

Duration of test:

until day 20 of gestation

Remarks:

Doses / Concentrations:
300, 1000, 5000 mg/kg bw/day (0.24, 0.8 and 4 % butan-1-ol in water)
Basis:
nominal in water

No. of animals per sex per dose:

11-17 females per dose

Control animals:

yes

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The general behaviour of the animals was observed throughout the experiment.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weight gain was monitored every week in the nonpregnant females and on days 3, 7, 10 and 17 of gestation in the pregnant animals

FOOD CONSUMPTION:
- The daily intake of food was monitored every week in the nonpregnant females and on days 3, 7, 10 and 17 of gestation in the pregnant animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The daily intake of water or n-butanol solutions was monitored every week in the nonpregnant females and on days 3, 7, 10 and 17 of gestation in the pregnant animals.

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- 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]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

In the case of variance homogeneity, one-way variance analysis and Dunnett tests were used; in the case of heterogeneity Kruskal-Wallis variance analysis was followed by non-parametric tests. Frequency data was analyzed with a Fisher exact probability test. The consumption of food and water or n-butanol solution, and body weight gain of dams were evaluated with two-way variance analysis and Scheffe test for multiple comparison.

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Details on maternal toxic effects:
The general appearance and behaviour of the animals exposed to n-butyl alcohol given in drinking water during the 8 weeks, as well as body weight gain, food and liquid intake were similar to that of the control animals . There were no cases of mortality in either group.
Integral toxicity indexes observed in the female rats, such as body weight gain during gestation, food and liquid (water or butanol solutions) intake, absolute and relative organ weights, hemoglobin concentration and haematocrit values did not differ between the exposed and control groups.

Dose descriptor:

NOAEL

Effect level:

5 000 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
The unit of statistical analysis in this study was the individual foetus, not the litter.
At 5000 mg/kg bw the crown-rump length was decreased (mean of 4.0 to 3.8 cm for the control and treated group, respectively) . Developmental effects were reported in all 3 dose groups. Skeletal effects were limited to an extra 14th rib in 1 foetus in the low dose group and 2 foetuses in the high dose group, and wavy ribs in 1 foetus in the low dose group. CNS defects included dilation of either the subarachnoid space or lateral and/or third ventricles of the brain, or external or internal hydrocephalus. Dilated renal pelves were also observed. Of the 65 control foetuses examined for skeletal effects, none had an extra fourteenth or wavy rib(s) or any other skeletal malformation or variation. Two of the 61 control foetuses examined for visceral anomalies had dilatation of the lateral and/or third ventricles of the brain, while none had dilatation of the subarachnoid spaceor external or internal hydrocephalus. Although the authors considered all 3 dose levels as related to increased foetal effects when compared to controls, there was no dosedependent increase. However, since no foetus of the middle dose group was affected, and only 2 in the low dose, it appears probable that the observations in the low dose group were not treatment related.

Dose descriptor:

NOAEL

Effect level:

5 000 mg/kg bw/day (nominal)

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The unit of statistical analysis in this study was the individual foetus, not the litter.

The authors considered the recorded developmental effects (dilatation of the brain ventricles/spaces or renal pelvis, internal hydrocephalus, wavy or extra ribs) as being related to butan-1-ol and assessed these findings as variations or delayed development commonly seen in large historical control databases. Of significance, the incidence of all but one of the reported developmental effects in the actual control population was 0%. In the MARTA-MTA 1995 database, using Crl: CD BR rat, the incidence of "cerebral ventricle, enlargement" was 2%/foetus or 4.4%/litter, and the incidence of "renal pelvis, dilated" 0.95%/foetus or 5.2%/litter (Wise and Petrere, 1996). The "malformations" reported that were assessed as "variations" in other databases should be classified based on the incidence within the rat strain. The incidence of variations within the rat strain used in this study is unknown, since the authors used a rat strain common only to their laboratory in. The laboratory diet was also unique. Since the strain of rat and type and quality of diet can have profound effects on rates of variations and malformations, and since there is no historical database available for the strain tested, the term “variation" has to be assigned with reservation. However, the term may still be appropriate since the variations reported are also common in several rat strains frequently used in the. In fact, Nelson et al (1989) described some of these variations following inhalation exposure to butan-1-ol. It should not be surprising that high oral doses of butan-1 -ol that alter normal maternal physiology would also cause an increase in common variations in laboratory rodents. Thus, the developmental effects seen by Sitarek et al (1994) cannot be regarded as a selective foetal effect.

 

Effect on developmental toxicity: via inhalation route

Dose descriptor:

NOAEC

1 000 mg/m³

Additional information

There are no data available on the developmental toxicity of the dibutyl esters of adipic and glutaric acid. However data exist for the methyl esters of these acids (reaction mass, and dimethyl glutarate on its own). An OECD reproductive/developmental screen is available for dibutyl adipate and there are developmental toxicity studies available on the dicarboxyllic acids and butanol (the metabolites of the REACH substance following hydrolysis of the esters).

Data from Dimethyl esters:

The effects of dibasic esters on prenatal development were assessed by the inhalation route in a teratogenicity study (equivalent to an OECD 414 study) in pregnant rats, exposed 6 hours per day to to the est concentrations of 0, 160, 400 or 1000 mg/m3 between gestation days 7 through 16. No relevant effects occurred in the number of resorptions, fetal weight and there were no significant differences in the incidence of external, visceral or skeletal abnormalities between groups including controls. The NOEC for teratogenicity was therefore 1000 mg/m3.

Data on dimethyl glutarate:

No developmental toxicity was observed following inhalation exposure to concentrations up to 1000 mg/m3 of dimethyl glutarate. The NOEC for developmental toxicity was therefore the top dose.

Data on dibutyl adipate:

There was no evidence of developmental toxicity (within the bounds of the parameters assessed) following dosing of dibutyl adipate prior to, during, and for 4 days after pregnancy in rats.

Data on Glutaric acid (Pentanedioic acid; CAS No. 110-94-1):

In a developmental study in rats groups of pregnant female CD rats were administered doses of glutaric acid a 0, 125, 400 and 1300 mg/kgbw/ day via gavage on gestation days 6 – 15; animals were sacrificed on gestation day 20 and examined for any gross pathological changes. At 400 mg/kg-bw/day and higher, clinical signs such as salivation, rales and nasal discharge were observed. Mean body weight gain was decreased at 1300 mg/kg -bw/day during the dosing period, but they were comparable to control animals during post-dosing (GD 15 – 20). These effects could be attributed to the acidity of the substance rather than evidence of systemic toxicity. At 1300 mg/kg-bw/day, one female died and one was sacrificed on gestation day 13. No changes were seen in reproductive status. There were no effects on the number of corpora lutea, implantation sites, number of live and dead fetuses or fetal body weights. No differences were seen in visceral and skeletal abnormalities between the control and the treatment groups. No adverse effects on pregnancy and no teratogenic effects were observed.

In a developmental study in rabbits, groups of pregnant female New Zealand White rabbits were administered glutaric acid at 0, 50, 160 and 500 mg/kg -bw/day via gavage on days 6 – 18 of gestation; females were sacrificed on gestation day 29 and examined for gross pathological changes. No changes were seen in reproductive status. There were no effects on the number of corpora lutea, implantations, number of live and dead fetuses, resorptions, fetal body weights or external, visceral and skeletal abnormalities. There were no adverse effects on pregnancy and no embryotoxic or teratogenic effects were observed.

Data on Adipic acid (Hexanedioic acid; CAS No. 124-04-9)

In a developmental study in rats, groups of pregnant female Wistar rats were administered adipic acid via gavage at 0, 2.9, 13, 62 and 288 mg/kg - bw/day during gestation days 6 – 15. Females were sacrificed on gestation day 20 and were examined for any gross pathological changes. There were no effects on the number of corpora lutea, implantations, number of live and dead fetuses, resorptions, fetal body weights, or external, visceral and skeletal abnormalities.

In a second study using rabbits, groups of pregnant female Dutch-belted rabbits were administered adipic acid at 0, 2.5, 12, 54 and 250 mg/kgbw/ day via gavage on gestation days 6-18; females were sacrificed on gestation day 29 and examined for gross pathological changes. No changes were seen in reproductive status. There were no effects on the number of corpora lutea, implantations, number of live and dead fetuses, resorptions, fetal body weights, and external, visceral and skeletal abnormalities. There were no adverse effects on pregnancy and no embryotoxic or teratogenic effects were observed.

Data on n-butanol

Results from valid experimental studies showed no indication, that Butan-1-ol caused fetotoxic or teratogenic effects in doses below maternal toxic doses in rats.

In a GLP conform study according to a Japanese guideline, 20 pregnant rats per dose were given drinking water containing 1-butanol at 0.2%, 1.0% or 5.0% (corresponding to ca. 316, 1454 or 5654 mg/kg/day) on days 0 to 20 of pregnancy (Ema et al. 2005). The rats were sacrificed on day 20 of pregnancy and both the dams and fetuses were examined. A significant decrease in maternal body weight gain accompanied by reduced food and water consumption was found at 5.0%. No significant increase in the incidence of pre- and postimplantation embryonic loss was observed in any groups treated with 1-butanol. Fetal weight was significantly lowered at 5.0%. Although a significant increase in the incidence of fetuses with skeletal variations and decreased degree of ossification was found at 5.0%, no increase in the incidence of fetuses with external, skeletal and internal abnormalities was detected in any groups treated with 1-butanol. The data demonstrate that 1-butanol is fetotoxic only at maternal toxic doses. No evidence for teratogenicity of 1-butanol was noted. Based on the significant decreases in maternal body weight gain and fetal weight, it is concluded that the no observed adverse effect levels (NOAELs) of 1-butanol for both dams and fetuses are 1.0% (1454 mg/kg/day) in rats.

In another study, groups of 15-18 female Sprague-Dawley rats were exposed at 8000, 6000, 3500, or 0 ppm (24.7, 18.5 or 10.8 mg/L) 1-butanol, for 7 hr/day on Gestation Days 1- 19 (sperm detection = day 0; Nelson et al. 1989). Dams were sacrificed on Gestation Day 20, and fetuses were individually weighed, tagged, and examined for external malformations. One-half of the fetuses were stained and examined for skeletal abnormalities, and the other half were examined for visceral defects using the technique. 8000 ppm produced narcosis in approximately one-half of the dams. Two of eighteen dams at 8000 ppm died during the exposure period. Food consumption was decreased in the 6000 and 8000 ppm exposed dams. Foetal weights were slightly decreased at 6000 and 8000 ppm groups. External foetal malformations were not observed. There were no differences in malformation rates (skeletal or visceral) or in rates of commonly observed variations. However, there was a slight increase in the percent of fetuses with any skeletal variation or malformation (mainly rudimentary cervical ribs) in the 8000 ppm group but not in the lower two exposure groups. The NOAEL for teratogenicity is 24.7 mg/L and the NOAELs for dams and fetuses are 10.8 mg/L.

In the study of Sitarek et al., there was a slight increase in the percent of fetuses with any skeletal variation or malformation (mainly rudimentary cervical ribs) in the 8000 ppm group but not in the lower two exposure groups .The interpretation of the results is difficult. The unit of statistical analysis in this study was the individual foetus, not the litter. The authors considered the recorded developmental effects (dilatation of the brain ventricles/spaces or renal pelvis, internal hydrocephalus, wavy or extra ribs) as being related to butan-1-ol and assessed these findings as variations or delayed development commonly seen in large historical control databases. Of significance, the incidence of all but one of the reported developmental effects in the actual control population was 0%. In the MARTA-MTA 1995 database, using Crl:CD BR rat, the incidence of "cerebral ventricle, enlargement" was 2%/foetus or 4.4%/litter, and the incidence of "renal pelvis, dilated" 0.95%/foetus or 5.2%/litter (Wise and Petrere, 1996) . The "malformations" reported that were assessed as "variations" in other databases should be classified based on the incidence within the rat strain. The incidence of variations within the rat strain used in this study is unknown, since the authors used a rat strain common only to their laboratory. The laboratory diet was also unique. Since the strain of rat and type and quality of diet can have profound effects on rates of variations and malformations, and since there is no historical database available for the strain tested, the term "variation" has to be assigned with reservation. However, the term may still be appropriate since the variations reported are also common in several frequently used rat strains. In fact, Nelson et al (1989) described some of these variations following inhalation exposure to butan-1-ol. It should not be surprising that high oral doses of butan-1-ol that alter normal maternal physiology would also cause an increase in common variations in laboratory rodents. Thus, the developmental effects seen by Sitarek et al (1994) cannot be regarded as a selective foetal effect. The NOAEL for maternal toxicity and teratogenicity is considered to be >= 5000 mg/kg bw.

In a behavioural teratogenicity study (Nelson et al. 1989), there were no behavioural teratogenic effects found in rats in doses up to 18.5 mg/L (6000 ppm).

Considering the overall weight of evidence the reaction mass of dibutyl adipate and dibutyl glutarate would not be developmentally toxic.

Justification for classification or non-classification

Based on the absence of any relevant sign of reproductive or developmental toxicity in the various studies available on the constituents or analogues of the REACH substance, and taking account of the classification criteria of Annex VI Directive 67/548/EEC or UN/EU GHS, no classification is warranted for reproductive and developmental toxicity.