Diisononyl adipate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) adipate

EC Number:

203-090-1

EC Name:

Bis(2-ethylhexyl) adipate

Cas Number:

103-23-1

Molecular formula:

C22H42O4

IUPAC Name:

bis(2-ethylhexyl) adipate

Test material form:

liquid

Specific details on test material used for the study:

Purity: 99.2% w/w
Batch number: Y02259/003/001
Stability under test conditions: 34 days at room temperature in the diet

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Specific Pathogen Free (SPF) colony at the Alderley Park Breeding Unit, ICI
- Age at study initiation: 28 days
- Weight at study initiation: (P) Males: 72.5 g; Females: 71.1 g
- Housing: 2 females or 1 male per cage
- Diet (e.g. ad libitum): CTI diet supplied by Special Diets Servies Limited
- Water (e.g. ad libitum): filtered tap water
- Acclimatisation period: 6-7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21
- Humidity (%): 45-60
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 3-4 August 1987 To: 12 January 1988

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food):
Dose level 300 ppm: 9.07g/30 kg
Dose level 1800 ppm: 54.44g/30 kg
Dose level 12000 ppm: 362.90g/30 kg

Details on mating procedure:

- M/F ratio per cage: 1 male and 2 female per cage
- Length of cohabitation: 10 days
- Proof of pregnancy: vaginal smear were examined daily
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): separately

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean concentrations within 2% of target concentration for all groups. DEHA was not detected in any control diet (detection limit 10ppm). Chemical stability of DEHA in diet was determined on three batches of diet at nominally 300 ppm and 12000 ppm. Satisfactory chemical stability was established.
Homogeneity of DEHA in diet mixtures was satisfactorily demonstraded on the first diet batch at nominally 300 and 12000 ppm DEHA.

Duration of treatment / exposure:

Males: 10 weeks premating + mating period
Females: 10 weeks premating, mating, gestation (app. 22 days), lactation (22 days)

Frequency of treatment:

The rats in each generation were fed experimental diets continuously until termination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm (nominal)

Remarks:

ca. 28 mg/kg bw (nominal in diet)

Doses / concentrations 2

Dose / conc.:

1 800 ppm (nominal)

Remarks:

ca. 170 mg/kg bw (nominal in diet)

Doses / concentrations 3

Dose / conc.:

12 000 ppm (nominal)

Remarks:

ca. 1080 mg/kg bw (nominal in diet)

No. of animals per sex per dose:

30 females and 15 males per group in total 4 groups.

Control animals:

yes, plain diet

Details on study design:

The dose leveis for this study were based on data from the literature (NTP, 1982) and included an anticipated no effect level and a level at which toxic effects of DEHA were expected at some stage during the course of the study.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes, at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes, once weekly

BODY WEIGHT: Yes, of all rats were recored at weekly intervals throughout the premating period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, each cage of rats was recorded throughout the premating periods and calculated on a weekly basis. The food utilisation value per cage was calculated as the weight gained by the animals in the cage per 100g of food eaten.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
A count of all live and dead pups was made within 24 hrs (day 1), at days 5, 11, 22, 29 and 36 post partum. The sexes of the pups were also recorded at these times.

Postmortem examinations (parental animals):

SACRIFICE
All animals at scheduled kills and those killed during the study were anaesthetised by inhalation of halothane BP vapour and killed by exsanguination. All surviving males were killed after completion of mating. All females were killed after weaning thier litters.

Histological examination: Cervix, Epididymis, Liver, Mammary gland, Ovary, Prostate, Seminal vesicle, Testis, Uterus, Abnormal tissues.

Postmortem examinations (offspring):

All pups were killed as soon as possible after Day 36 post partum.

Statistics:

Mean bodyweight gain, food consumption and food utilisation during the premating period, female bodyweight gain during pregnancy, parental liver weights and pup (litter) bodyweight gain until Day 36 post partum.

Reproductive indices:

Mean lenght of gestation, mean pre-coital interval

Offspring viability indices:

Mean live born index, mean survival index, mean litter size, total litter weight and whole litter losses.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related abnormalities

BODY WEIGHT
Bodyweight gain was marginally reduced for high dose females. The difference was statistically significant during pregnancy weeks 2 (-15%) and 3 (-10%). Body weight was also significantly reduced (-6%) at the end of pregnancy (intermediate data not given). Body weight for parental females was not included in the study report for the lactation period. There was no effect on body weight gain in any other treatment group.

TEST SUBSTANCE INTAKE
There was a slight increase in food consumption in males dosed with 12000ppm DEHA from 6-10 weeks of the study, the effect being statistically significant at weeks 6-9. Food utilisation was slifghtly less efficients overall for males receiving 12000ppm DEHA.

REPRODUCTIVE PERFORMANCE
There was no effect on male or female fertility, gestation length, and pre-coital interval in any dose group. Litter size was slightly and not significantly reduced in the high dose group, but because of the minimal difference and the fact, that the number of live born pubs was unaffected by treatment, this effect is considered incidental.

ORGAN WEIGHTS
An increase in absolute (+ app. 18%) and relative (+18.9% males, 19.7% females) liver weight was observed for animals receiving 12000ppm DEHA. No other groupp treatment group was effected. This increase in liver weight has been reported previously and is associated with peroxisome proliferation (Moody and Reddy 1978).

GROSS PATHOLOGY
No treatment related abnormalities, with the possible exception of an accentuated lobular pattern in the liver of two high dose females.

HISTOPATHOLOGY
No treatment related abnormalities were observed. This includes, that no microscopic changes were detected in the reproductive tract of animals which failed to breed successfully, to account for suspected infertility.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

ca. 1 080 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

other: There was no effect on the fertility of male or female animals even at the highest dose.

2

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

ca. 170 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There were 4 whole litter losses. None in the control group, one in the 300ppm DEHA dose group, two in the 1800ppm DEHA dose group, and one
in the 12000ppm DEHA dose group. These were of a low lncidence, not dose related, and therefore not related to treatment with DEHA.

CLINICAL SIGNS (OFFSPRING)
No treatment related effect

BODY WEIGHT
There was no difference in pub mean weight on PND1, but mean pup weight gain and consequently total litter weight for high dose male and female offspring were reduced throughout the whole of the post partum phase. There was no effect on either male or female pup weight gain in any other dose group in comparison with the control animals.

GROSS PATHOLOGY (OFFSPRING)
No treatment related effect

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 170 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

In this study 15 male and 30 female Wistar rats per group were fed DEHA in the diet from 10 weeks before mating and through mating (males) or until day 22 postpartum (females) at levels of 28, 170 and 1080 mg/kg bw/day (300, 1800 or 12 000 ppm). At 1080 mg/kg, body weight gain was marginally reduced in females during premating. This difference was statistically significant during gestation weeks two and three and led to significant lower body weight (-6%) at the end of gestation. No body weight was reported for the lactation period. Liver weights of both male and female parental animals were significantly increased, most likely due to peroxisiome proliferation as has already been reported in the literature (Moody D E, Reddy J K (1978). Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol Appl Pharmacol 45 (2) 497-504).

There were no effects on male or female fertility, gestation length, and pre-coital interval up to the highest dose level (1080 mg/kg), which also led to reduced body weight gain in the parental generation, reduced total litter weights, and reduced body weight gain in the pups. There was also a minimal and not statistically significant decrease in litter size in this group, but since there was no difference in the total number of pups born and pup survival, this observation was considered incidental and not toxicologically relevant. No differences in clinical signs or treatment-related macroscopic abnormalities were found in the pups of all groups.

More than 90% of 30 females treated for 10 weeks with up to 1080mg/kg became pregnant within 5 days, and no difference in the pre-coital interval was observed compared to control animals.

The systemic NOAEL for both generations was conservatively set to 170mg/kg because of the reduced body weight and / or body weight gain in parental females and offspring and the increase in parental liver weights in high dose animals observed at the next higher dose of 1080mg/kg, even though the effects on pups are probably secondary to maternal toxicity. Since reproductive performance was unaffected by treatment, the NOAEL for fertility is 1080mg/kg.