Reaction mass of diisobutyl adipate and diisobutyl glutarate and diisobutyl succinate

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 diisobutyl esters of adipic, succinic and glutaric acid. However data exist for the methyl esters of these acids, isobutanol and dibutyl adipate (a structurally similar analogue to one of the components). Dosing of the diisobutyl esters will result in the release of the acids and isobutanol, therefore read across to the dimethyl esters is considered apropriate since the major hydrolysis product of the dimethyl esters is the acids. In support of this, data on isobutanol are also provided to address the isobutanol that would be released from the diisobutyl esters once entering the body.

In developmental toxicity studies, no effects were observed on mating performance, fertility, gestation duration, litter size, development or viability, and lactation performance in rats by inhalation.

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:

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 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).

Reference 3

Endpoint:

two-generation reproductive 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:

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:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: (P) ca. 7 wks; (F1) ca. 4 wks
- Weight at study initiation: (P) Males: 236-350 g; Females: 159-213 g; (F1) Males: post natal day 32: means: 84-97 g; Females: post natal day 32: means: 78-88 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 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
Exposures were conducted in four 2.0 m3 stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. Treatment of exhaust air consisted of charcoal- and HEPAfiltration. The generation system was operated as follows: Vapors of the test article were generated using a heated bead column. The test article was introduced to the top of the column, while nitrogen entering the bottom of the column served as the carrier gas. For Groups 2 and 3 the column was 2.4 cm in diameter (ID), 40 cm long filled with 2, 3 and 4 mm beads. For Group 4, the column was 5 cm in diameter (ID) and 68 cm long filled with 3, 6, 8 and 12-mm beads. The columns were wrapped with heating tapes (Omega Engineering). Temperatures were set to approximately 100°C for chambers 2 and 3, and approximately 170-185ºC for chamber 4 using an Omega CN370 temperature controller. The chamber 4 temperature was significantly higher due to the need to maintain an internal temperature of at least 70ºC to ensure vaporization of the test article. The test article was vaporized as it dripped from 1/16-inch Teflon tubing onto the glass beads contained within each heated column. The test article was metered from an amber glass reservoir to the column using an FMI pump (Fluid Metering, Inc., Oyster Bay, New York). Calibrated FMI pumps included 2 model no. QG-6 pumps with a 1/4-inch piston for chamber 2, and a 3/8-inch piston for chamber 3 and a model no. QG-20 pump with a 1/4-inch piston for chamber 4. Vaporization nitrogen was delivered from the facility nitrogen generation system and was controlled using calibrated flowmeters (Gilmont Instruments, Barrington, Illinois). The vaporization nitrogen carried the isobutanol vapor through Teflon delivery lines (3/8-inch O.D. tubing for chambers 2 and 3, 1-inch O.D. tubing for chamber 4) to the chamber inlet where the concentration was reduced to the desired level with chamber ventilation air. The animal exposure was initiated by switching the FMI pumps and the
compressed nitrogen on simultaneously.

TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least one standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored continuously and were recorded approximately every 35 minutes. Oxygen content within the chamber was measured during the pre-study method development phase. Nominal chamber concentrations were determined daily. Total air volume was calculated by multiplying mean chamber ventilation rate (in liters per minute) by the exposure duration (in minutes). Test atmosphere homogeneity was demonstrated during pre-study method development. There were no detectable aerosols at any evaluation interval.

- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation:
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing the female was placed into a plastic maternity cage with nesting material (also no evidence of mating was apparent)
- After successful mating each pregnant female was caged: in a plastic maternity cage with nesting material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.

Duration of treatment / exposure:

Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
7 days/week prior to mating, during mating and gestation; treatment was suspended during lactation days 0-4 and re-initiated on lactation day 5

Frequency of treatment:

daily for 6 hours per day

Details on study schedule:

- F1 parental animals not mated until 11-12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 15-16 weeks

Remarks:

Doses / Concentrations:
0, 500, 1000, 2500 ppm (ca. 1.48, 2.95 and 7.37 mg/l)
Basis:
nominal conc.

No. of animals per sex per dose:

30

Control animals:

yes

Details on study design:

- Dose selection rationale: Exposure levels were selected based upon a reduction in response to external stimuli (during exposure) observed in a
previous 90-day neurotoxicity study and a non-statistically significant reduction in body weights and body weight changes during
postnatal days 4-7 and 7-14 in pups from dams exposed to 2500 ppm isobutanol in a probe study.

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
Detailed physical examinations were recorded weekly for all parental animals throughout
the study period. All animals were observed twice daily for moribundity and mortality; in
addition, the animals were observed for appearance, behavior and pharmacotoxic signs
within one hour after completion of exposure. Females expected to deliver were also
observed twice daily during the period of expected parturition and at parturition for
dystocia (prolonged labor, delayed labor) or other difficulties.
During exposure, special attention was given to the state of arousal and response to novel
stimuli by producing a loud-noise stimulus (only for individual animals visible in the
chamber). The noise was produced by allowing a ¾-inch PVC pipe coupling
(approximately 50 g) to strike the glass of the exposure chamber at the approximate level
of the cage rack. The coupling was attached to a length of cotton rope that was held
against the glass approximately 60 cm from the coupling. The coupling was raised until
the rope was approximately perpendicular to the glass, and the coupling was released.
The response was recorded as: 1) no reaction to stimulus, 2) slight reaction, ear flick or
some evidence that stimulus was heard, or 3) more energetic response (jump, flinch, may
include vocalization). Due to the cage rotation procedure, approximately 10-30% of the
animals could be visible on a given day.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual F0 and F1 male body weights were recorded on study days 0, 1, 4 and 7, weekly
thereafter throughout the study, and prior to the scheduled necropsy. Individual F0 and F1
female body weights were recorded on study days 0, 1, 4 and 7 and weekly thereafter
until evidence of copulation was observed. Mean weekly body weights and body weight
changes are presented for each interval. Once evidence of mating was observed, female
body weights were recorded on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days
1, 4, 7, 14 and 21; for the F0 females, body weights were also recorded on lactation day
28. Body weight changes are presented for each of these intervals. After weaning
(lactation day 28), weekly body weights were recorded for these F0 females until the
scheduled necropsy.


FOOD CONSUMPTION::
Individual F0 and F1 male and female food consumption was measured on study days 0, 1,
4 and 7 and weekly thereafter until pairing. Food intake was not recorded during the
mating period. Male food consumption was measured after mating on a weekly basis
until the scheduled necropsy. Female food consumption was recorded on gestation
days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21; for the F0 females, food
consumption was also recorded on lactation day 28. For the F0 generation, the last
scheduled interval for weekly recording of food consumption was study day 126. Since
final body weights and clinical observations were recorded on the scheduled necropsy
days (after study day 126), food consumption was manually recorded at that time. These
data are not presented in the report tables, but will be maintained in the raw data. Food
consumption was calculated and reported as g/animal/day and g/kg/day for the
corresponding body weight change intervals. Food efficiency (body weight gained as a
percentage of food consumed) was also calculated and reported for these intervals.

Oestrous cyclicity (parental animals):

Vaginal smears were prepared daily to determine the stage of estrus for each female,
beginning 21 days prior to pairing and continuing until evidence of mating was observed.
For females with no evidence of mating, smearing was continued until termination of the
mating period. The average cycle length was calculated for complete estrous cycles (i.e.,
the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus
[P] beginning 21 days prior to initiation of the mating period and until the detection of
evidence of mating). Estrous cycle length was determined by counting the number of
days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle
during which evidence of mating was observed for a given animal was not included in the
mean individual estrous cycle length calculation. Vaginal smears were also performed on
the day of necropsy to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
[testis weight, epididymis weight, sperm production rate, sperm count in left testis, sperm count in left epididymis, sperm motility, sperm morphology]

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 / F2] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]


GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving adults were euthanized following the selection of the F1 generation and
completion of a detailed clinical observation.
- Maternal animals: All surviving adults were euthanized following the selection of the F1 generation and
completion of a detailed clinical observation.


GROSS NECROPSY
- The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the
thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
organs collected:
Adrenals (2); Aorta; Bone with marrow (sternebrae); Brain (forebrain, midbrain, hindbrain); Coagulating gland; Eyes with optic nerve (2);
Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum); Heart; Kidneys (2); Liver (sections of two lobes);
Lungs (including bronchi, fixed by inflation with fixative); Lymph node (mesenteric); Ovaries and oviducts (2); Pancreas; Peripheral nerve (sciatic);
Pituitary; Prostate; Salivary gland [submandibular (2)]; Seminal vesicles (2); Skeletal muscle (rectus femoris); Skin with mammary gland;
Spinal cord (cervical); Spleen; Testes with epididymidesa (the right testis and epididymis were fixed in Bouin's solution) and vas deferens; Thymus;
Thyroids [with parathyroids, if present (2)]; Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions
organ weights:
Adrenals; Brain; Epididymides (total and caudal; these paired organs were weighed seperately); Kidneys; Liver; Ovaries; Pituitary; Prostate; Seminal
vesicles with coagulating glands (with accessory fluids), Spleen, Testes (these paired organs were weighed seperately), Thymus gland; Uterus with
oviducts and cervix
organs examined:
Adrenal glands: cortex and medulla; Brain; Cervix; Epididymis (right): caput, corpus and cauda; Kidneys; Liver; Ovaries; Pituitary, Prostate; Seminal
vesivles with coagulatin glands (with accessory fluids); Spleen; Testis (right); Thymus, Uterus (with oviducts); Vagina; All gross (internal) lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [28 (F1) and 21 (F2)] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Prior to weaning, 30 F1 pups/sex/group were randomly selected for the F1 parental
generation and for evaluation of developmental landmarks. Additional F1 pups were
retained as potential replacement animals, and were euthanized by CO2 inhalation and
necropsied on PND 34, 36 or 37. In addition, one F1 and one F2 pup/sex/litter (when
available) were selected from the F1 and F2 weanlings for complete necropsy on PND 28
and PND 21, respectively; brain, spleen and thymus gland weights were recorded. All
remaining non-selected F1 and F2 weanlings were euthanized by CO2 inhalation and
necropsied on PND 28 and PND 21, respectively, with emphasis on developmental and
reproductive system morphology. All gross lesions from F1 and F2 weanlings were
preserved in 10% neutral-buffered formalin for possible future histopathologic
examination; all other tissues were discarded.


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


HISTOPATHOLOGY / ORGAN WEIGTHS
- All gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.

Statistics:

detailed statistical analyses were performed (see other information on materials and methods)

Reproductive indices:

Mating and fertility indices were calculated:
Male (Female) Mating Index (%) = [No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for
Mating] x 100
Female Fertility Index (%) = [No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating] x 100
Male Fertility Index (%) = [No. of Males Siring a Litter / Total No. of Males Used for Mating] x 100

Offspring viability indices:

survival indices were calculated (not further specified)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
After 91 days of exposure to the test article, female no. 74805 in the 500 ppm group had
pale eyes and ears and appeared to be having difficulty during parturition (gestation day
23) and was euthanized in extremis on lactation day 0. This female delivered 12 pups and
had four pups retained in utero. On the day of euthanasia, this female had pale ears and
eyes and red discharge from the vagina. Microscopically, the cause of death for this
female was determined to be severe acute renal tubular necrosis and moderate acute
hepatic necrosis. Therefore, this death was not attributed to the test article; no evidence
of dystocia was observed in females in the 1000 or 2500 ppm groups. All other animals
survived to the scheduled necropsy.
There were no test article-related clinical observations at the weekly detailed physical
examinations or one hour following exposure. The response to novel stimulus was
similar in all groups, including the control group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean F0 body weights, body weight gains and cumulative body weight gains in the 500,
1000 and 2500 ppm groups were unaffected by exposure to the test article during the premating
period and one week following weaning (females) and throughout the study
(males). The only statistically significant (p<0.05) difference from the control group was
a decreased mean body weight gain in the 1000 ppm group males during study days
84-91. A similar reduction was not observed in the 2500 ppm group; therefore, this
transient decrease was not attributed to the test article.
No test article-related effects on maternal body weights or body weight gains were
observed in the test article-exposed groups during the F0 gestation period. Differences
from the control group were slight and were not statistically significant.
No test article-related effects on F0 lactation body weights or body weight gains were
observed in the test article-exposed groups. The only statistically significant (p<0.01)
differences from the control group were a mean body weight loss of 33 grams in the
500 ppm group compared to a loss of 53 grams in the control group during lactation days
21-28, resulting in a statistically significant (p<0.05 or p<0.01) increase in mean body
weight on lactation day 28 and in mean body weight gain during the entire lactation
period (days 1-28). Since the control group lost more weight than the 500 ppm group,
this difference was not considered to be an adverse change.
Food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the
500, 1000 and 2500 ppm groups were unaffected by exposure to the test article during the
pre-mating period and one week following weaning (females) and throughout the study
(males). Occasional statistically significant (p<0.05 or p<0.01) reductions in food
consumption (primarily g/kg/day) were observed in the 2500 ppm group males compared
to the control group values during study days 14-21 through 112-119. Food consumption
(g/animal/day) in these males was generally not affected during these intervals. Mean
body weights and food efficiency in the 2500 ppm group males and females were similar
to or greater than the control values. Therefore, these sporadic reductions were not
attributed to exposure to the test article. The only other statistically significant
differences (p<0.05 or p<0.01) from the control group were a decrease in food
consumption (g/animal/day) in the 2500 ppm group males during study days 28-35, an
increase in food consumption (g/kg/day) during day 0-1 in the 1000 ppm group males and
a decrease in food efficiency in the 1000 ppm group males during study days 1-4. Similar
differences were not observed in the 2500 ppm group males during this interval;
therefore, no relationship to exposure was evident.
F0 maternal food consumption and food efficiency during gestation were unaffected by
test article exposure in the 500, 1000 and 2500 ppm groups. The only statistically
significant difference from the control group was a slight reduction in food consumption
(g/kg/day) in the 2500 ppm group when the entire gestation period (days 0-20) was
evaluated. Food efficiency and gestation body weight gain in this group was unaffected
during this interval. Therefore, the reduction was not attributed to test article exposure.
F0 maternal food consumption and food efficiency in the test article-exposed groups were
similar to the control group values throughout lactation. The only statistically significant
(p<0.01) differences were a smaller decrement in food efficiency in the 500 ppm group
during lactation days 21-28 than the control group and an increase in food efficiency in
this group during the entire lactation period (days 1-28). Because similar effects were not
observed at higher exposure levels, these differences were not considered test articlerelated.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The mean lengths of estrous cycles in the test groups were similar to that in the control group. None of the differences were statistically
significant.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No test article-related effects were observed on F0 spermatogenesis endpoints (mean
testicular and epididymal sperm numbers, sperm production rate, motility, progressive
motility and morphology) in males at any exposure level. Differences from the control
group were slight and were not statistically significant. A slight reduction in the
percentage of morphologically normal sperm in the 2500 ppm group (95.9%) compared
to the control group value (97.9%) was attributed to a single male (no. 74628) with an
abnormally low percentage of morphologically normal sperm (0.5%). The values for the
remaining males in this group were comparable to those in the control group. Therefore,
the reduction in morphologically normal sperm in the single male in the 2500 ppm group
was attributed to biological variation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test article-related effects on reproductive performance were observed. Male and
female mating indices were 86.7%, 100.0%, 96.7% and 100.0% in the control, 500, 1000,
and 2500 ppm groups, respectively. Male and female fertility indices were 73.3%,
96.7%, 93.3% and 93.3% in the same respective groups. The only statistically significant
(p<0.05) differences from the control group were increases in the male and female
fertility indices in the 500 ppm group. It should be noted that the fertility index for the
control group (73.3%) was reduced compared to the mean value in the WIL historical
control data (90.4%). However, the values in the isobutanol-exposed groups exceeded
the mean value in the WIL historical control data. Males that did not sire a litter
numbered 8, 1, 2 and 2 in the control, 500, 1000 and 2500 ppm groups, respectively.
Females that had evidence of mating but did not deliver numbered 4, 1, 2 and 3 in the
same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups
were similar to the control group value. The mean lengths of estrous cycles in these
groups were similar to that in the control group. None of the differences were statistically
significant.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test article-related effects on organ weights were observed in the 500, 1000 and
2500 ppm group males or females. The only statistically significant (p<0.05 or p<0.01)
differences from the control group were increases in mean absolute prostate weights in
the 1000 ppm group males and mean relative prostate weights in the 500 and 1000 ppm
group males and a decrease in mean absolute pituitary weight in the 500 ppm group
females. Because similar effects were not observed in the 2500 ppm group males and
females, these changes were not attributed to the test article.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Female no. 74805 in the 500 ppm group was euthanized on lactation day 0 due to
difficulties during parturition. This female delivered 12 pups and had four fetuses with
no apparent malformations retained in utero. This animal had dark red contents in the
ileum and jejunum and a pale pituitary gland. Similar findings were not observed in
females at the scheduled necropsy in the higher exposure groups. Therefore, these
findings were not attributed to the test article.
At the scheduled necropsy, no exposure-related trends were observed in the macroscopic
findings noted in the test article-exposed groups. Findings were observed similarly in
control group animals, were noted infrequently and/or did not occur in an
exposure-related manner. The mean number of implantation sites and the mean number
of unaccounted sites in the F0 test article-exposed females were similar to the control
group values; no statistically significant differences were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Female no. 74805 was euthanized in extremis during parturition. Microscopic findings
for this female included severe acute renal tubular necrosis, moderate acute hepatic
necrosis, lymphoid depletion of the spleen and lymphoid necrosis of the thymus. The
cause of the moribund condition of this female was renal and liver necrosis with multiple
organ failure.
At the scheduled necropsy, no test article-related microscopic findings were observed
including for animals that failed to breed or produce a litter. The only statistically
significant (p<0.05) differences from the control group values were an increase in the
incidence of hydronephrosis in the 500 ppm group females and a decrease in the
incidence of dilatation of the uterine lumen in the 2500 ppm group females. A slightly
increased incidence (not statistically significant) of basophilic tubules was observed in the
kidneys of the 2500 ppm group males; however, the severity of this lesion was minimal,
and a similar increase was not observed in the 2500 ppm group females. Therefore, this
common, spontaneous alteration was not considered to be test article-related. Other
microscopic findings were typical of spontaneous conditions in young rats.
OTHER FINDINGS (PARENTAL ANIMALS)
GESTATION LENGTH AND PARTURITION:
Mean F0 gestation lengths in the test article-exposed groups were unaffected by exposure
to the test article. Differences from the control group value were slight and were not
statistically significant. The mean gestation lengths in the 500, 1000 and 2500 ppm
groups were 21.9, 21.9 and 21.8 days, respectively, compared to a mean gestation length
of 22.0 days in the control group.
Female no. 74805 in the 500 ppm group was euthanized in extremis due to apparent
difficulty during parturition. The cause of moribundity for this animal was determined to
be renal and liver necrosis with multiple organ failure. Because dystocia was not
observed in females in the 1000 and 2500 ppm groups, the difficulty during delivery was
not attributed to the test article.

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: (ca. 7.37 mg/l)

VIABILITY (OFFSPRING)
F1:
The mean numbers of F1 pups born, live litter sizes and percentages of males per litter
were similar among all groups; no statistically significant differences were noted.
Postnatal survival on PND 0 (relative to the number born) and during PND 0-1, 1-4, 4-7,
7-14, 14-21, 21-28, birth to PND 4 and PND 4-28 were unaffected by test article
exposure. Statistically significant (p<0.05 or p<0.01) decreases in postnatal survival were
observed in the test article-exposed groups relative to the control group values. These
reductions occurred in the 500 ppm group during PND 0-1, 4-7 and 4-28 and in the
1000 ppm group during PND 0-1. Reductions (not statistically significant) were observed
in the 2500 ppm group during PND 0-1, birth to PND 4 and PND 4-28. Because no
exposure concentration-dependent response was observed, these decreases in postnatal
survival were not considered test article-related.
F2:
Postnatal survival, the mean number of pups born, the percentage of males at birth and
live litter size on PND 0 in the 500, 1000 and 2500 ppm groups were unaffected by
exposure to the test article. Differences from the control group were slight and were not
statistically significant.

CLINICAL SIGNS (OFFSPRING)
F1:
The numbers of pups (litters) available for clinical signs were 299 (22), 390 (29),
390 (27) and 388 (27) in the control, 500, 1000 and 2500 ppm groups, respectively. The
numbers of pups found dead during the lactation period were 13, 22, 21 and 30 in the
control, 500, 1000 and 2500 ppm groups, respectively. In the same respective exposure
groups, 3, 19, 9 and 11 pups were missing and presumed cannibalized. The incidence of
dead/missing pups was slightly higher in the 2500 ppm group, although this was not
considered exposure-related. In general, larger-sized litters tend to have increased
postnatal deaths due to competition with littermates for access to maternal resources
(nursing, thermoregulation, grooming, etc.). The dams in the 2500 ppm group had
approximately one additional pup/litter compared to the control group. The pups from
female no. 74805 (500 ppm group) were euthanized on PND 0 due to the euthanasia of
the dam. The general physical condition of the F1 pups during lactation was generally
similar in all groups, including the control group.
All F1 adults survived to the scheduled necropsy. There were no test article-related
clinical observations at the detailed physical examinations or one hour following
exposure. The response to novel stimulus in the test article-exposed groups was similar
to that in the control group.
F2:
The numbers of pups (litters) litters available for clinical signs were 286 (22), 348 (25),
316 (23) and 334 (24) for the control, 500, 1000 and 2500 ppm groups, respectively. The
numbers of pups found dead over the entire lactation period numbered 6, 11, 10 and 10 in
the control, 500, 1000 and 2500 ppm groups, respectively. In the same respective
exposure groups, 1, 4, 4 and 1 pups were missing and presumed cannibalized. The
general condition of the pups in the test article exposure groups was similar to that in the
control group; findings were noted infrequently and/or did not occur in an exposure
concentration-related manner.

BODY WEIGHT (OFFSPRING)
F1:
No exposure-related effects on mean pup body weights or body weight gains were
observed during the pre-weaning period (PND 1-28). Numerous statistically significant
(p<0.05 or p<0.01) decreases in mean pup body weights and/or pup body weight gains
were observed in the 500 and 1000 ppm group F1 males and females, primarily during
PND 4-28. Mean pup body weights in the test article-exposed groups were similar to the
control group value on PND 1. Mean pup body weights in the 2500 ppm group were also
reduced (not statistically significant) between PND 14 and 28 (6.4%-8.1% for the males
and 4.9%-7.7% or the females). Mean pup body weight gains in the 2500 ppm group
were similar to those in the control group during PND 1-4, 4-7 and 7-14. Mean pup body
weight gains in this group were slightly reduced during PND 14-21 and 21-28
(statistically significant at p<0.05 during PND 14-21, males only). These reductions in
mean body weights and mean body weight gains followed neither a
concentration-dependent nor a time-course response. The earliest, most severe reductions
were observed in the 500 ppm group and began on PND 4, which was prior to the
re-initiation of maternal exposure and continued throughout the pre-weaning periods.
The onset of reductions in mean body weight in the 1000 ppm group occurred, at a lesser
extent, on PND 14; statistical significance in this group was not observed after PND 21.
There were no reductions in mean body weight observed during the pre-weaning period in
the 2500 ppm group; only slight reductions (statistically significant only in males at one
time point) in mean body weight gains were observed during the last half of the
pre-weaning period. Because of the lack of an exposure-related effect, none of the
decreases were attributed to the test article.
No exposure-related effects on mean pup body weights or body weight gains were
observed during the post-weaning period (PND 28-32 and 32-35). Statistically
significant (p<0.05 or p<0.01) decreases in mean pup body weights and/or pup body
weight gains were observed in the 500 and 1000 ppm group F1 males and females during
this period. The reductions did not occur in a concentration-dependent manner; therefore,
the decreases were not attributed to the test article. Mean pup body weights and body
weight gains in the 2500 ppm group were similar to those in the control group. No
statistically significant differences were noted.
No test article-related trends in F1 mean body weights, body weight gains or cumulative
body weight gains were observed in the test article-exposed groups. Numerous
statistically significant (p<0.05 or p<0.01) differences (primarily decreases) in mean body
weights and body weight gains were observed in males and females in the test
article-exposed groups. The majority of the differences were observed in the 500 and
1000 ppm group males. Mean body weights in these males were 10.3% and 9.7%,
respectively, lower than the control group value at the end of the pre-mating period (study
day 204). These decrements appeared to be a continuation of the decreases in mean body
weight gain observed in the 500 and 1000 ppm groups during the pre-weaning and early
post-weaning periods (refer to Section 6.1.8.3). The effects were most pronounced at the
low-exposure level, less pronounced at the mid-exposure level and not expressed at the
high-exposure level. Because the changes were not concentration-dependent, they were
not attributed to exposure to the test article.
No test article-related differences in mean maternal body weights or body weight gains
were observed in the F1 females during gestation. Differences from the control group
values were slight, and none were statistically significant.
No test article-related differences in mean maternal body weights or body weight gains
were observed in the F1 females during lactation. Differences from the control group
values were slight, and none were statistically significant.
F2:
No test article-related effects were observed on mean F2 pup body weights or body weight
gains. The F2 control group litters were slightly smaller by approximately one pup than
the 500 (PND 1 only), 1000 and 2500 ppm group litters (PND 1 and 4); however, the
statistical analyses on mean body weight and body weight gains were performed using the
litter size as the covariant. The F2 control group pups generally weighed more than the F1
control group pups in this study and the pups in the WIL historical control data for
inhalation studies.
The slightly increased litter sizes (0.8-1.1 pups/dam) in the 1000 and 2500 ppm groups
correlated with concomitant reductions in F2 mean pup body weights (7% and 3%,
respectively, in the males; 7% and 4%, respectively, in the females) on PND 1. Mean pup
F2 body weights in the 500 ppm group males and females were 7% and 9%, respectively,
lower than the concurrent control group values on PND 1. The differences in the 500 and
1000 ppm groups were statistically significant (p<0.05) compared to the control group
values. However, the F2 mean male and female pup weights in the concurrent control
group on PND 1 were 4%-6% greater than the mean male and female pup weights in the
inhalation studies in the WIL historical control data and 6% greater than the concurrent
control group pups in the F1 generation in this study. Mean body weight gain in the 1000
and 2500 ppm group males and females were similar to the control group values during
PND 1-4. Mean body weight gains in the 500 ppm group males and females were
decreased (statistically significant at p<0.05) compared to the control group values during
PND 1-4. On PND 4, mean male pup body weights were 12%, 8% and 1% lower than
the concurrent control group value in the 500, 1000 and 2500 ppm groups, respectively;
mean female pup weights were 12%, 7% and 2% lower in the same respective groups.
The differences in both genders were statistically significant (p<0.01) only in the
500 ppm group. However, the concurrent F2 control group male and female mean body
weights were 8% greater than those in the WIL historical control data for inhalation
studies and 11% and 9% greater than the mean F1 male and female pup weights,
respectively, on PND 4. Due to the lack of effects in the 1000 and 2500 ppm groups, the
apparent influence of litter size on mean pup weights and the difference of the concurrent
control group values compared to the historical control data for inhalation studies, the
differences in mean body weights and body weight gains in the F2 pups during PND 1-4
were not attributed to test article exposure.
Following standardization of litters (to eight pups/dam) on PND 4, mean male and female
pup weights in the 500 ppm group were 17% and 15%, respectively, lower than the
concurrent control group values (statistically significant at p<0.01) on PND 7. Mean
male and female pup weights in the 1000 ppm group were 9% lower than the concurrent
control group values; the difference was statistically significant (p<0.05) only for the
females. Mean pup body weights in the 2500 ppm group were unaffected by F1 maternal
exposure on PND 7. Mean body weights gains in the 500 and 1000 ppm groups were
reduced during PND 4-7. The differences from the control group were statistically
significant (p<0.01) only for the 500 ppm group males and females. Mean body weight
gains in the 2500 ppm group males and females was slightly (not statistically significant)
less than those in the concurrent control group, but consistent with the calculated gain in
the WIL historical control data for inhalation studies. The mean body weight gains in the
1000 and 2500 ppm groups were comparable to those in the F1 control group. Due to the
lack of effect at the highest exposure concentration and the difference of the concurrent
control group values from the historical control values, these differences in F2 postnatal
body weights and body weight gains were not considered exposure-related.
On PND 14, mean male and female pup body weights in the 500 ppm group were 11%
and 10%, respectively, lower than the concurrent control group values; the differences
were statistically significant (p<0.05 or p<0.01). The mean body weights for the 1000
and 2500 ppm group males and females were similar to the historical control values for
inhalation studies, but were lower (5%-8%) than the concurrent control group values (not
statistically significant). In addition, the PND 14 mean body weights for the 500, 1000
and 2500 ppm group males and females were comparable to or greater than the values for
the F1 control group pups. Mean body weight gains in the 500, 1000 and 2500 ppm group
males and females were slightly reduced (not statistically significant) compared to the
concurrent control group values during PND 7-14. The concurrent control group values
during this interval were 3% higher than the calculated mean gain in the WIL historical
control data for inhalation studies. In addition, mean F2 body weight gains in all groups
during PND 7-14 were equal to or higher than the gain in the F1 control group pups at this
same interval. Due to the lack of effect in the 1000 and 2500 ppm groups and the
difference of the concurrent F2 control group values compared to the F1 control group
values, the differences in mean body weights on PND 14 was not considered
exposure-related.
On PND 21, mean male and female pups body weights in the 500, 1000 and 2500 ppm
groups were reduced (statistically significant at p<0.05 or p<0.01) compared to the
concurrent control group values. However, the F2 control group values on this day were
14% higher than the F1 pups on PND 21 and 5% higher than the mean body weight on
PND 21 in the WIL historical control data for inhalation studies. The 2500 ppm group
values were higher than the F1 control group values, while the 500 and 1000 ppm group
values were lower than the F1 control group and the inhalation historical control group
values. Mean body weight gains in the 500, 1000 and 2500 ppm group males and
females were also reduced (statistically significant at p<0.01) compared to the concurrent
control group value. Mean body weight gains in these groups were also reduced
compared to the F1 control group value and the calculated value in the WIL historical
control data. However, the most severe reductions occurred in the 1000 ppm group males
and females and the least severe reductions were observed in the 2500 ppm group males
and females. Therefore, these reductions were not considered exposure-related due to the
lack of an exposure-response relationship.

SEXUAL MATURATION (OFFSPRING)
F1:
There were no test article-related differences in the mean day of acquisition of
balanopreputial separation or mean body weight on the day of acquisition. The only
statistically significant difference (p<0.05) from the control group was a reduced mean
body weight on the day of balanopreputial separation in the 500 ppm group. However,
because similar decreases were not observed in the 1000 and 2500 ppm groups, the
decrease was not attributed to the test article.
There were no test article-related differences in the mean day of acquisition of vaginal
patency or mean body weight on the day of acquisition. The only statistically significant
difference (p<0.05) from the control group was a delay in the day of acquisition of
vaginal patency in the 500 ppm group. Similar effects were not observed in the 1000 and
2500 ppm groups; therefore, the delay was not attributed to the test article.
Reproductive performance was unaffected by test article exposure at concentrations of
500, 1000 and 2500 ppm.
F1 male and female fertility indices were 73.3%, 83.3%, 76.7% and 80.0% in the control,
500, 1000 and 2500 ppm groups, respectively. Male and female mating indices in the
same respective groups were 96.7%, 93.3%, 90.0% and 83.3%. None of the differences
from the control group were statistically significant. Males that did not sire a litter
numbered 8, 5, 7 and 6 in the control, 500, 1000 and 2500 ppm groups, respectively.
Females that had evidence of mating but did not deliver numbered 7, 3, 4 and 1 in the
same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups
were similar to the control group value. The mean lengths of estrous cycles in these
groups were similar to that in the control group. None of the differences were statistically
significant.
No test article-related effects on F1 spermatogenic endpoints (mean testicular and
epididymal sperm numbers, sperm production rate, sperm motility, progressive motility
and the percentage of morphologically normal sperm) were observed at any exposure
level. Differences from the control group were slight and were not statistically
significant.

ORGAN WEIGHTS (OFFSPRING)
F1:
No test article-related changes in mean organ weights (absolute or relative to final body
weight) were observed in F1 pups in the 500, 1000 or 2500 ppm groups. The only
statistically significant (p<0.05) differences from the control group were decreases in the
mean absolute thymus weight in the 500 ppm group males and the mean absolute brain
weight in the 500 ppm group females. Mean body weights on PND 28 was reduced in the
500 ppm group males and females. In addition, similar decreases were not observed in
the 1000 or 2500 ppm groups nor in the relative organ weights in the 500 ppm group
animals; therefore, these reductions were not attributed to the test article.

No test article-related changes in mean organ weights (absolute or relative to final body
weight) of the F1 adults were observed. The only statistically significant (p<0.05 or p<0.1)
differences from the control group values were reductions in mean absolute liver weight in the
500 ppm group males and mean absolute pituitary weight in the 500 and 1000 ppm group
females, and increases in the mean relative (to final body weight) thymus and brain
weights in the 500 ppm group males. Mean final body weights in these groups were
reduced compared to the control group values, resulting in reduced absolute weights for
the liver and pituitary. Because the brain is unaffected by reductions in body weight,
increased mean relative brain weights would be expected. Similar changes were not
observed in the 2500 ppm group males and females. Therefore, these changes were not
attributed to the test article.
F2:
No test article-related effects on mean F2 pup organ weights (absolute and relative to final
body weight) were observed. Occasional statistically significant (p<0.05 or p<0.01)
differences from the control group were noted in the 500, 1000 and 2500 ppm groups, but
no exposure-related trends were evident.

GROSS PATHOLOGY (OFFSPRING)
F1:
There were no test article-related internal findings noted at the necropsies of pups that
were found dead. Pup no. 74782-14 in the 500 ppm group had dark red discoloration of
the brain. Pup no. 74782-08 in the same group had lungs that were not fully collapsed
(PND 29). In the 2500 ppm group, pup no. 74803-14 had a mechanical injury (fractured
right parietal bone with associated hemorrhage). Aside from the presence or absence of
milk in the stomach, no other internal findings were noted in pups that died.
No test article-related internal findings were observed in the weanlings necropsied on
PND 28, 34, 36 or 37, or in the pups euthanized due to the death of the dam. Pup
no. 74785-10 in the 500 ppm group had a raised, white area on the median lobe of the
liver. Pup nos. 74739-03 and 74849-12 in the 1000 ppm group had a dilated renal pelvis
(unilateral). In the 2500 ppm group, pup no. 74813-14 had a cystic oviduct. Pup
no. 74862-12 in the same group had a mechanical injury (the frontal and parietal bones
were fractured). No other internal findings were observed.
No internal findings were observed at the necropsy of pups selected for organ weights.
No test article-related internal findings were observed at the scheduled necropsy of F1
adult males and females. Findings observed were noted similarly in the control group,
did not occur in an exposure concentration-related manner and/or were common to
laboratory rats. The mean numbers of implantation sites and the numbers of unaccounted
sites in the test article-exposed groups were similar to the control group values.
F2:
No test article-related internal findings were observed in F2 pups that were found dead.
Pup no. 1 from the litter of dam no. 74789-09 in the control group had a mechanical
injury consisting of fractured pubis, ischium and ilium bones and all sacral vertebrae.
Pup no. 1 from the litter of dam no. 74748-11 in the 1000 ppm group had dark red
discoloration of all lobes of the lungs. Aside from the presence or absence of milk in the
stomach, no other internal findings were observed.
No test article-related internal findings were observed in the F2 weanlings not selected for
organ weights. Two pups each in the 500 and 1000 ppm groups had a dilated renal pelvis
(unilateral). No other internal findings were observed.
No test article-related internal findings were observed in the F2 weanlings selected for
organ weights. One pup in the 2500 ppm group had a dilated renal pelvis (unilateral). No
other internal findings were observed.

HISTOPATHOLOGY (OFFSPRING)
F1:
There were no test article-related histopathologic alterations observed in F1 animals at
exposure levels of 500, 1000 and 2500 ppm, including for animals that failed to breed or
produce a litter. Findings in the test article-exposed groups occurred similarly in the
control group and were considered spontaneous or incidental in nature. The only
statistically significant (p<0.05) difference from the control group was a decrease in the
number of males in the 2500 ppm group with subacute inflammation in the kidneys. A
decrease in the number of animals with microscopic lesions is not considered adverse.
Therefore, these differences were not considered test article-related. No test
article-related effects on primordial follicle counts or corpora lutea counts were observed
in the 2500 ppm group females.

OTHER FINDINGS (OFFSPRING)
FOOD CONSUMPTION:
No test article-related trends in F1 food consumption (g/animal/day and g/kg/day) or food
efficiency were observed in the test article-exposed groups. Occasional statistically
significant (p<0.05 or p<0.01) differences from the control group were noted, but no test
article-related trends were apparent. Statistically significant (p<0.05 or p<0.01) decreases
in food consumption evaluated as g/animal/day were observed in the 500 and 1000 ppm
group males. Because the changes observed in the test article-exposed groups were not
concentration-dependent, there were no exposure-related changes in food efficiency and
food consumption in the males, evaluated as g/kg/day, was generally similar to that in the
control group, the decreases were not attributed to test article exposure. These reductions
in food consumption (g/animal/day) were attributed to the smaller size of the males in
these groups. In the 2500 ppm group males and females, food consumption
(g/animal/day and/or g/kg/day) was occasionally reduced (statistically significant at
p<0.05 or p<0.01) compared to the control group values. Specifically, food consumption
(g/kg/day) in the 2500 ppm group females was generally reduced throughout the
pre-mating period and also during the post-mating period (study days 260-266). The
differences from the control group were often statistically significant (p<0.05 or p<0.01).
However, food efficiency in the 2500 ppm group males and females was similar to that in
the control group and no trends were apparent that would be consistent with a test
article-related effect.
F1 maternal food consumption and food efficiency during gestation in the test article
exposure groups was unaffected by test article exposure. The only statistically significant
(p<0.05 or p<0.01) differences from the control group were reductions in food
consumption (evaluated as g/kg/day) in the 2500 ppm group during gestation days 0-4
and 11-14. Because similar decreases were not observed in the g/animal/day values, they
were not attributed to exposure.
F1 maternal food consumption and food efficiency in the test article exposure groups were
unaffected by test article exposure during lactation. The only statistically significant
(p<0.05 or p<0.01) differences from the control group were reductions in food
consumption (evaluated as g/animal/day and/or g/kg/day) in the 500, 1000 and 2500 ppm
groups during lactation days 14-21. Because of the absence of a concentration-dependent
response, these decreases were not attributed to test article exposure.
GESTATION LENGTH AND PARTURITION:
F1 maternal food consumption and food efficiency in the test article exposure groups were
unaffected by test article exposure during lactation. The only statistically significant
(p<0.05 or p<0.01) differences from the control group were reductions in food
consumption (evaluated as g/animal/day and/or g/kg/day) in the 500, 1000 and 2500 ppm
groups during lactation days 14-21. Because of the absence of a concentration-dependent
response, these decreases were not attributed to test article exposure.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: (ca. 7.37 mg/l)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 500 ppm

Sex:

male/female

Basis for effect level:

other: (ca. 7.37 mg/l)

Reproductive effects observed:

not specified

F0 and F1 parental survival were unaffected by test article exposure in all exposure
groups. No test article-related effects were observed on F0 and F1 reproductive
performance (estrous cycles, mating and fertility indices, the mean number of days
between pairing and coitus and the mean length of gestation), body weights, food
consumption and food efficiency in the 500, 1000 and 2500 ppm group males or females.
Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm
production rate, motility, progressive motility and the percentage of morphologically
normal sperm) in the F0 and F1 test article-exposed groups were unaffected by test article
exposure. There were no test article-related macroscopic findings or changes in mean
organ weights in the F0 or F1 males and females in the 500, 1000 and 2500 ppm groups.
Microscopic evaluation of the F0 and F1 males and females revealed no test article-related
histopathologic lesions, including for animals that failed to breed or produce a litter. No
test article-related effects on primordial follicle counts and corpora lutea counts were
observed in the F1 2500 ppm group females. F1 and F2 pup survival and the general
physical condition of the pups were unaffected by exposure to the test article. No test
article-related effects on mean pup body weights were observed in the 500, 1000 or
2500 ppm group F1 or F2 pups. No test article-related macroscopic findings were
observed in F1 or F2 pups that were found dead or euthanized at the scheduled necropsy.
There were no test article-related changes in mean organ weights for the F1 or F2 pups.
The mean day of acquisition of balanopreputial separation or vaginal patency and mean
body weights on the day of acquisition in the F1 test article-exposed pups were not
affected by the test article.

Reference 4

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study planned (based on read-across)

Justification for type of information:

There are no data available on the reproductive toxicity of the reaction mass of bis(2-methylpropyl) pentanedioate and bis(2-methylpropyl) butanedioate and bis(2-methylpropyl) hexanedioatethe (also called "diisobutyl esters of adipic, succinic and glutaric acid", abbreviated as DBE-IB). However a one-generation reprotox study data exist for the methyl esters of these acids (DBE). In this study rats were exposed to the test concentrations of 0, 160, 400 or 1000 mg/m3 for 14 weeks pre-breeding, 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.

Following a compliance check of the dossier on DBE, ECHA has not accepted the above study to fulfil the requirements of endpoint 7.8.1 in the DBE dossier and has issued a draft decision (Communication number: CCH-D-2114331899-34-01/D, dated 20 July 2016) to the lead registrant, pursuant to Article 41(1) and (3) of the REACH Regulation, to submit the following information: Extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443), in rats, oral route, according to the following study-design specifications:
- At least two weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation

As Chemoxy International s a co-registrant of methyl esters of adipic, succinic and glutaric acid (DBE), it proposes to use the requested EOGRTS with DBE as read-across to fulfill the requirements of REACH with respect to endpoint 7.8.1 for DBE-IB. Metabolism of the diisobutyl esters will result in the release of the acids and isobutanol, therefore read across to the dimethyl esters is considered appropriate since the major hydrolysis products of the dimethyl esters are the corresponding acids. Isobutanol would be released from the diisobutyl esters once entering the body. In the available 2-generation study with isobutanol in rats, there were no reported effects on fertility or toxicity to the offspring in either generation at any dose. As such, isobutanol is not considered to be a reproductive toxicant under the circumstances of this study.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Justification for study design:

The basic test design of an extended one-generation reproductive toxicity study (test method EU B.56./OECD TG 443 with Cohorts 1A and 1B, without extension of Cohort 1B to include a F2 generation, and without Cohorts 2A, 2B and 3) is a standard information requirement as laid down in column 1 of 8.7.3., Annex X. The extended one-generation reproductive toxicity study would meet the information requirement of Annex X, Section 8.7.3 of the REACH legislation.

Species:

rat

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available on the reproductive toxicity of the diisobutyl esters of adipic, succinic and glutaric acid (DBE-IB). However data exist for the methyl esters of these acids (DBE), isobutanol and dibutyl adipate (DBA, a structurally similar analogue to one of the components). Dosing of the diisobutyl esters will result in the release of the acids and isobutanol, therefore read across to the dimethyl esters is considered appropriate since the major hydrolysis products of the dimethyl esters are the corresponding acids. In support of this, data on isobutanol are also provided as isobutanol would be released from the diisobutyl esters once entering the body.

 

Data on the Dimethyl Esters:

The effects of dibasic esters (DBE) on fertility were assessed by the inhalation route in a One-Generation study (equivalent to an OECD 415 study) in rats, exposed to the test concentrations of 0, 160, 400 or 1000 mg/m3 for 14 weeks pre-breeding, 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 (DBA):

In an OECD 421, doses of up to 1000 mg/kg bw/day had no effect on any reproductive or developmental parameter assessed in the study. As such the NOEL for reproductive toxicity is considered to be 1000 mg/kg bw/day.

 

Data on Isobutanol:

In the available 2-generation study in rats there were no reported effects on fertility or toxicity to the offspring in either generation at any dose. As such, isobutanol is not considered to be a reproductive toxicant under the circumstances of this study.

 

Taking both sets of data together it is concluded that the diisobutyl esters of adipic, succinic and glutaric acid would not be expected to be toxic to reproduction. As such it is considered highly unlikely that extending dosing of the materials above out to a second generation would have identified any reproductive toxicity not observed in the single generation study.

Effects on developmental toxicity

Description of key information

No effects were observed on number of resorptions or fetal weight and no increase in external, visceral or skeletal abnormalities in offspring from parent rats exposed by inhalation.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no data available on the developmental toxicity of the diisobutyl esters of adipic, succinic and glutaric acid (DBE-IB). However data exist for the methyl esters of these acids (DBE), and for isobutanol. Dosing of the diisobutyl esters will result in the release of the acids and isobutanol, therefore read across to the dimethyl esters is considered appropriate since the major hydrolysis product of the dimethyl esters is the acids. In support of this, data on isobutanol are also provided to address the isobutanol that would be released from the diisobutyl esters once entering the body.

 

Data from Dimethyl Esters:

The effects of dibasic esters (DBE) and dimethyl glutarate (as such) on prenatal development were assessed by the inhalation route in teratogenicity studies (equivalent to an OECD 414 study) in pregnant rats, exposed 6 hours per day to the test concentrations of 0, 160, 400 or 1000 mg/m3 between gestation days 7 through 16. No relevant effects occurred in the number of resorptions, foetal weight and there was 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 from Isobutanol:

In both the rat and rabbit, dosing of isobutanol produced no evidence of developmental toxicity at any dose. In the rabbit there was a small decrease in maternal bodyweight in the highest dose but this was not sufficient to consider it a significant adverse treatment related effect.

Taking into account the data on the dimethyl esters and the isobutanol data it is concluded that the diisobutyl esters 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 rats up to the concentration of 1000 mg/m3 by inhalation exposure, 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.