COASOL 290 PLUS

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dimethyl esters of adipic, glutaric and succinic acids

IUPAC Name:

Reaction mass of dimethyl esters of adipic, glutaric and succinic acids

Details on test material:

see information in the field 'Confidential details on test material'

Test animals

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

Administration / exposure

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;

Doses / concentrations

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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:

2

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).