Diisononyl adipate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

As no data were available on diisononyl adipate (DINA), data on the structural analogue diethylhexyladipate were used.
One-generation reproduction study in rats: NOAEL (fertility): 1080 mg/kg bw; no effects on fertility (CEFIC, 1988, comparable to OECD guideline 415)

Link to relevant study records

Reference

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

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

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

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

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.

Dose / conc.:

300 ppm (nominal)

Remarks:

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

Dose / conc.:

1 800 ppm (nominal)

Remarks:

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

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

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.

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.

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.

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

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

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

Critical effects observed:

not specified

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 080 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The one-generation study (CEFIC, 1988) was considered key, as it was a GLP study performed equivalent to OECD Guideline 415. 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.

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.

In a 28 -day repeated dose study by Miyata et al. in 2006, test substance administration had no effect on sperm parameters, hormonal status, and histopathology of reproductive organs up to 1000mg/kg with the exception of 4 of 10 high dose females, which showed increased ovarian follicle atresia. Two of these females had a prolonged estrous cycle (4 and 10 days, until sacrifice), although no changes in hormones were detected. In the above cited one-generation study, 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. In this study as well as in a chronic study (NTP, 1982) using 50 animals per sex and group at doses up to 1250mg/kg no increase in ovarian follicle atresia was reported as well as no changes in further reproductive organs (Cervix, Epididymis, Mammary gland, Ovary, Prostate, Seminal vesicle, Testis, Uterus). Thus, since the effects observed with only 10 animals after 4 weeks could not be confirmed by two studies using higher animal numbers, which were treated with comparable doses for significantly longer time periods, it is concluded that DEHA does not adversely affect fertility.

No hints for estrogenic activity of DEHA were detected in an immature type uterotrophic assay performed by the Korean Food and Drug Administration (Park et al., 2007, see IUCLID section 7.9.3). No changes in absolute organ weights, serum FSH and LH levels, or uterine morphological changes such as luminal epithelial height, myometrial thickness, numbers of uterine glands and BrDU indices were observed up to a dose of 1000mg/kg/day.

Effects on developmental toxicity

Description of key information

As no data were available on DINA, data on the structural analogue diethylhexyladipate were used. 
Rat, diet: NOAEL teratogenicity = 1080 mg/kg, NOAEL maternal tox. = 170mg/kg (CEFIC 1988, similar to OECD 414, treatment from gestation day 1 to 22, GLP)
Rabbit, diet: NOAEL teratognicity and maternal tox. = 145mg/kg (BASF 2014, OECD 414, GLP)
One-generation reproduction study in rats: reduced pub weights at 1080mg/kg, NOAEL  170mg/kg (CEFIC, 1988, comparable to OECD guideline 415)

Link to relevant study records

Reference

Endpoint:

developmental 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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

For determination of limit dose the OECD 414 guideline was followed. The bottom dose level was selected based on information obtained from literature and was related to likely human exposure.
The maximum human intake has been estimated by MAFF (UK) 1986 to be 16 mg/day and this was calculated to be 25 mg/kg/day for a 60-70 kg human. A factor of 100 was then used to provide an appropriate margin of safety which thus gave a dose of 25 mg/kg/day in rats for the present study. The middle dose was spaced between these two doses using approximately a sixfold factor. The dose levels were then calculated as ppm in the diet (for a 300g rat eating 25g food per day). The rats were dosed on Days 1-22 inclusive of gestation, Day 1 being the day that mating was confirmed by a sperm-positive vaginal smear.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity: 99.2% w/w
Supplier: ICI France, Department Baleycourt.
Appearance: colourless liquid.
Batch: Y02259/003/003-4
Chemical stability was proven at 300ppm and 12000 for 34 days, which is in excess of the maximum period of 21 days between fresh diet preparations.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test animals: Wistar rats of the Alpk: AP fSD strain (from the specific pathogen free (SPF) colony.
Initial weight: 218-278 g.

Housing:
Animal Breeding Unit and Experimental Unit, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.
For the duration of the study, each rat was individually housed in rat racks supplied by All Type Tools Ltd, Woolwich, London, UK . The cages had solid stainless steel sides and the floor, back and front wer e constructed of 14SWG stainless steel mesh . The internal measurements were 34 .0 x 37 .5 x 20 .3cm3,with a floor area of 1275cm2 . The cages were suspended over collecting trays lined with absorbent paper . On the front of each cage was a card identifying the animal by individual number, dose group and study . Tap water via an automatic watering system and food were available ad libitum.
The temperature of the animal room was within the range of 19-24° C (as recorded daily by a maximum and minimum thermometer) with a mean of 22°C . Relative humidity was within a recorded range of 44-70% (as assessed by daily readings from a hygrometer) and mean of 54% . There were at least 12 air changes per hour . The artificial lighting was controlled by a,time switch and provided alternate periods of 12 hours light and 12 hours darkness throughout the study.

Diet:
All diets were based on CT1 diet supplied by Special Diets Services Ltd, Witham, Essex, UK. The experimental diets were prepared in 30kg batches from premixes and dispensed into glass feeding jars . Two batches of diet were prepared at each level.

Diet sampling and analysis:
A sample was taken from each diet prepared . Samples were taken from the diet feeding jars and analysed. Chemical stability of DEHA in CT1 diet was determined at 300 and 12000ppm .
Homogeneity of DEHA was also examined in a concurrent study (Tinston 1988) and found to be satisfactory .

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From every prepared diet a sample was taken and analysed to verify the correct concentrations. Results were within 8% of the target concentration.

The amount of food consumed by each animal was measured daily by giving a weighed quantity of food contained in a glass jar on one day and calculating the amount consumed from the residue on the next day.

Details on mating procedure:

Wistar-derived, virgin female rats were paired overnight at the Breeding Unit with unrelated males of the same strain. On the following morning, vaginal smears from these females were examined for the presence of sperm. The day when spermatozoa were detected was designated Day 1 of gestation and on this same day, successfully mated females were delivered to the experimental unit at CTL . A total of 96 mated females was supplied over a two week period. On arrival, the rats were within the weight range 218-278g and were approximately 12 weeks of age. Twelve female rats were supplied on each of eight days.

Duration of treatment / exposure:

From Day 1 of gestation until termination on Day 22.

Frequency of treatment:

Each day

Duration of test:

The in life phase of the study was conducted from 15 September to 16 October 1987.

Dose / conc.:

300 ppm (nominal)

Remarks:

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

Dose / conc.:

1 800 ppm (nominal)

Remarks:

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

Dose / conc.:

12 000 ppm (nominal)

Remarks:

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

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

The study was divided into 24 replicates (randomised blocks) with each replicate containing one rat from each dosage group. Cages within the replicates were assigned to one of the four groups using computer-generated random number permutations. The individual animal numbers were then assigned sequentially within the relevant groups to give the rack plan. On arrival (Day 1 of gestation) each rat was allocated to a cage (and therefore a treatment group) randomly within the replicate and individually identified by ear punching with the number assigned to it from the experimental design. Replicates were filled sequentially with three replicates added to the study on each of the eight days on which rats were received .

Maternal examinations:

Clinical observations:
All animals were checked on arrival to ensure that they were physically normal externally . They were subsequently observed daily for any changes in behaviour or clinical condition and these were recorded.

Body weight:
The bodyweight of each animal was recorded daily on Days 1 to 22 inclusive of gestation .

Terminal Investigations:
On Day 22 of gestation all the animals were killed by over exposure to halothane BP (FLUOTHANE , ICI Pharmaceuticals, Macclesfield, Cheshire, UK) vapour . A post mortem was performed and all animals were examined macroscopically. The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination. The following data was recorded:
Number of corpora lutea in each ovary.
Number and position of implantations subdivided into:
(a) live foetuses.
(b) early intra-uterine deaths.
(c) late intra-uterine deaths.
Intra-uterine deaths were classified as follows:
Early intra-uterine deaths showed decidual or placental tissue only. Late intra-uterine deaths
showed embryonic or foetal tissue in addition to placental tissue. The implantations were assigned letters of the alphabet to identify their
position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.

Fetal examinations:

Each foetus was weighed and individually identified within the litter by means of a cardboard tag. After weighing, the foetuses were killed with an intra-cardiac injection of pentobarbitone,sodium solution, 200mg/ml, (EUTHATAL, May and Baker Ltd, Dagenham, Essex, UK).

Assessment of Teratogenicity:
Each foetus was examined for external abnormalities and for cleft palate. All foetuses were then examined internally for visceral abnormalities under magnification, sexed, eviscerated and fixed in methanol. The head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities. (The brains of one litter, female 72, 1800ppm, inadvertently were not examined.) The carcasses were then returned to methanol for subsequent processing and staining with Alizarin Red S. The stained foetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed and the result converted to a four point scale. Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external, visceral or skeletal examination) defects. Variations were also recorded and classified as minor defects or variants depending on the historical frequency of occurrence in rats of this strain.

Statistics:

The following data were considered by analysis of variance :
(i) Maternal bodyweight gain.
(ii) Maternal food consumption.
(iii) The numbers of implantations and live foetuses per female.
(iv) Percentage pre-implantation loss and percentage post-implantation loss (calculated on an individual litter basis). The percentage pre-implantation loss and post-implantation loss were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The analyses of variances were weighted by the denominator in the proportion.
(v) The percentage of implantations which were early intra-uterine deaths (calculated on an individual litter basis).
(vi) Gravid uterus weight, litter weight and mean foetal weight (calculated on an individual litter basis).
(vii) Mean manus and pes score per foetus (calculated on an individual litter basis).
(viii) The percentage of foetuses with minor external/visceral defects only, external/visceral variants and minor skeletal defects only (calculated on an individual litter basis).

The analyses of variance allowed for the replicate structure of the study design and were carried out using the GLM procedure in SAS (1985). Unbiased estimates of the treatment group means were provided by the least square means (LSMEANS option in SAS). Individual treatment group means were compared with the control group mean using Student's t-test based on the error mean square in the analysis.

Further analysis:
Fisher's Exact Test, comparing each treated group with the control group.

All statistical tests were one-sided with the following exceptions which were two-sided: maternal bodyweight gain, maternal food consumption and the proportion of male foetuses.

Details on maternal toxic effects:

Maternal toxic effects:yes.
Remark: reduced body weight gain and food consumption

Details on maternal toxic effects:
Administration of 12000 ppm DEHA resulted in slight maternal toxicity (small but significant maternal reduction in body weight gain (-13%) and food consumption). At 1800 ppm DEHA, there was no evidence of maternal toxicity.

No treatment related clinical signs, mortality or macroscopic anomalies were observed.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: Based on effects at 12000 ppm (1080 mg/kg).

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes.
Remark: reduced ossification and increase in the incidence of visceral variants, not considered adverse

Details on embryotoxic / teratogenic effects:
Administration of 1800 and 12000ppm DEHA resulted in minimal foetotoxicity (reduced ossification and increase in the incidence of visceral variants, when compared to control groups), which were not considered adverse. Incidences of slightly dilated ureter were significantly increased in the high dose group compared to concurrent control values, but were well within historical control data. Though an increase in kinked ureters was proposed by the authors, the increase was slight, not significant, and within historical control data.

A dietary level of 300 ppm DEHA was a clear no-effect level for embryonic development. There was no effect on foetal weight, litter weight, gravid uterus weight, number of intra-uterine deaths, or number of external abnormalities. At the highest dose, there was a minimal and not significant decrease in litter size (10.7 vs. 11.8), but the change was too small to be of toxicological significance. No historical control data on litter size was given. Additionally, there was no effect on number of corpora lutea, implantations, and post-implantation loss.

Dose descriptor:

NOAEL

Effect level:

ca. 1 080 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

ca. 1 080 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Dose descriptor:

NOEL

Effect level:

28 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

170 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Diet analysis: chemical stability of DEHA in diet was satisfactory.

Concentrations of DEHA were within acceptable limits.

Clinical observations: All rats survived to scheduled termination.

The clinical findings observed were considered not to be related to DEHA administration.

Maternal body weight: Administration of 12000 ppm DEHA was associated with a small but statistically significant reduction in bodyweight gain compared with the control group which was most marked at the start of the feeding period.

Maternal food consumption: Maternal food consumption was statistically significantly reduced in the 12000ppm group from Days 2-18 inclusive of pregnancy. There were no adverse effects on food consumption in the 300 or 1800 ppm DEHA groups.

Maternal macroscopic findings (post mortem): macroscopic changes were considered not to be related to DEHA treatment.

There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external

abnormalities.

The incidence of minor external and visceral defects was unaffected by treatment although two visceral variants were increased at the top two dose levels; kinked ureter being slightly and not significantly increased in the 1800 and 12000 ppm groups and slightly dilated ureter being increased in the 12000 ppm group. Incidences for both effects were within historical control data for this laboratory. Overall, minor skeletal defects were increased in a dose-related manner at 1800 and 12000 ppm DEHA, while skeletal variants and pes score were increased at the top dose only. These findings indicate slightly poorer ossification at the 1800 and 12000 ppm dose levels. The reduced ossification are considered to be the result of slight foetotoxicity. There was no treatment-related effect on skeletal or visceral variants at 300 ppm DEHA.

Executive summary:

24 pregnant Wistar rats were exposed via the diet to on average 28, 170, and 1080mg/kg from gestation day 1 to 22 in a study following GLP criteria and similar to OECD guideline 414 (CEFIC, 1988). Administration of 1080mg/kg resulted in slight maternal toxicity, expressed as a significant reduction in body weight gain and a slight, but also significant reduction in food consumption. There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external abnormalities. The incidence of minor external and visceral defects was unaffected by treatment although two visceral variants were increased at the top two dose levels, kinked ureter being increased in the 1800 and 12000 ppm groups, though not statistically significant, and slightly dilated ureter being increased in the 12000 ppm group. Incidences for both effects were within historical control data and are thus not considered adverse. Findings indicate dose-dependent slightly poorer ossification at the 1800 and 12000 ppm dose levels. The reduced ossification are not considered adverse, but as the result of minimal foetotoxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

170 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no studies available for diisononyl adipate, but two prenatal developmental toxicity studies were performed with DEHA in rats and rabbits.

In a study according to GLP and OECD 414 pregnant rabbits were treated with DEHA in the diet at 40, 80 and 160 mg/kg bw/day, which corresponded to 36, 70, and 145mg/kg/day based on the average relative food consumption (BASF, 2014). The mean test article intake was highest at the beginning and exceeded the average value of 145mg/kg/day until day 18, which is the most sensitive period in fetal development in rabbits. No maternal toxicity was observed in all dose groups. 20 control and 19 litters per treatment group were available for evaluation. There was no difference between treated and control animals with regard to implantation loss, corpora lutea, litter size, sex distribution, fetal body weight, and external and visceral malformations or variations. Doses were based on a preliminary range finding study, in which 5 mated females were exposed to 100, 300, and 1000mg/kg from days 7 -29 post coitum. Severe toxicity (e.g., body weight loss) was noted after treatment with 300mg/kg in the diet and via gavage (to exclude palatability problems), so that the maximum dose in the main study was app. one half of 300mg/kg.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 160mg/kg bw/dayin the diet (dose level approximation). Based on the average relative food consumption, this level corresponded to 145 mg/kg bw/day (BASF, 2014)

24 pregnant Wistar rats were exposed via the diet to on average 28, 170, and 1080mg/kg from gestation day 1 to 22 in a study following GLP criteria and similar to OECD guideline 414 (CEFIC, 1988). Administration of 1080mg/kg resulted in slight maternal toxicity, expressed as a significant reduction in body weight gain and a slight, but also significant reduction in food consumption. There was no effect at any dose on foetal weight, litter weight, gravid uterus weight, numbers of intra-uterine deaths or numbers of external abnormalities. The incidence of minor external and visceral defects was unaffected by treatment although two visceral variants were increased at the top two dose levels, kinked ureter being increased in the 1800 and 12000 ppm groups, though not statistically significant, and slightly dilated ureter being increased in the 12000 ppm group. Incidences for both effects were within historical control data and are thus not considered adverse. Findings indicate dose-dependent slightly poorer ossification at the 1800 and 12000 ppm dose levels. The reduced ossification are not considered adverse, but as the result of minimal foetotoxicity.

In a supporting publication by Singh et al only 5 rats per group were treated by intraperitoneal injection, a route which is not relevant, with up to 10ml/kg (9250mg/kg b.w.). No increase in skeletal malformations, visceral or gross abnormalities and no differences in the number of corpora lutea, resorptions, and live fetuses were observed. The two highest doses (5 and 10ml/kg) led to a slight but significant reduction in foetal body weight, but the relevance of this effect cannot be assessed, since no data on maternal toxicity was presented.

The NOAEL for developmental toxicity is based on the one-generation study, which showed reduced pup body weight gain and reduced total litter weight at 1080 mg/kg. Even though these effects were most likely secondary to maternal toxicity, the NOAEL for developmental toxicity was set to 170mg/kg, following the precautionary principle.

Justification for selection of Effect on developmental toxicity: via oral route:
In two OECD 414 studies in rats and rabbits, no adverse effects on the offspring were observed. But in the above mentioned one-generation study, pup body weight gain and total litter weight were reduced at 1080mg/kg. Even though these effects were most likely secondary to maternal toxicity, the NOAEL for developmental toxicity was set to 170mg/kg, following the precautionary principle.

Justification for classification or non-classification

The available developmental and one-generation reproduction toxicity data do not trigger classification for developmental toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008).