1,2-Benzenedicarboxylic acid, di-C16-18-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study with acceptable restrictions Not full study report published, but main information is given in sufficient details.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Principles of method if other than guideline:

Study contents reported here were chosen to determine whether exposure to D911P impairs reproductive performance in the rat, including sensitive periods of development.

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 (Margate, Kent, UK)
- Age at study initiation: (P) 6 wks; (F1) x wks
- Weight at study initiation: (P) Males: 145 to 228 g; Females: 115 to 198 g;
- Housing: Animals were housed in cages in groups of four of the same sex, except during mating when males and females were pair-housed, and during gestation and weaning when females were housed individually.
- Diet (ad libitum): LAD 2 SQC, Special Diet Services, Witham, UK
- Water: ad libitum
- Acclimation period: 13 days

Route of administration:

oral: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required dietary concentrations of both substances were achieved by direct dilution of a premix with untreated diet. All formulations were prepared on a weekly basis.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 3 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually.

Treatment of the F0 generation continued for 10 weeks before pairing. For the females, the duration and regularity of the oestrus cycle was established by daily vaginal smears, commencing 10 days before pairing. At pairing, one male and one female were housed together, taking care not to pair
siblings, for up to 3 weeks. Cages were examined each morning for evidence of copulation plugs and vaginal smears were collected for assessment of spermatozoa and stage of oestrus. Males that failed to mate were replaced with another of proven fertility in order to maximise the
number of litters. The day on which evidence of mating was found was designated Gestation Day (GD) 0, and females were then removed to individual cages. From GD 20, females were checked three times daily for evidence of onset, progress, and completion of parturition. All animals were
allowed to litter naturally (F0 offspring), and rear their own offspring until Day 21 of lactation. Dams were examined daily for evidence of normal maternal behaviour.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Tests indicated that the method of preparation produced a homogenous distribution of test substance, which was stable under the conditions used. Samples of diet were analysed for each group at weeks 1, 8 (control and 1.0% only), 11, 14, 18, 28, and 31. All formulations were within 8.5% of the nominal concentrations.

Duration of treatment / exposure:

Continuously over two generations.
Treatment of the F0 generation continued for 10 weeks before pairing.

Frequency of treatment:

ad libitum feeding

Details on study schedule:

- Selection of parents from F1 generation when pups were 25 days of age.
Animals of F1 generation were mated in the 10th week postweaning.

Remarks:

Doses / Concentrations:
0, 0.1, 0.5 and 1.0/2.0%
Basis:
nominal in diet
Highest dose was reduced to 1.0 % after week 7

Remarks:

Doses / Concentrations:
0,120,600 and 2400 mg/kg bw/d
Basis:
actual ingested
F0 males and females in the first week of treatment

Remarks:

Doses / Concentrations:
0, 61, 312, 684 and 0, 77, 388, 792 mg/kg bw/d
Basis:
actual ingested
in males and females after the 7th week

Remarks:

Doses / Concentrations:
0, 56, 280, 619 and 0, 63, 328, 696 mg/kg bw/d
Basis:
actual ingested
in males and females by 10th week

Remarks:

Doses / Concentrations:
0, 163, 867, 1760 mg/kg bw/d
Basis:
actual ingested
in females during lactation

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based on information for similar substances, a pilot study was conducted by using dietary inclusion levels of 0.1%, 0.5%, and 1.0%. There were no
apparent treatment-related effects on either the F0 or F1 generation in this study (not reported here). Therefore, dietary inclusion levels for the main study were set at 0.1%, 0.5% and 2.0% D911P. Because of marked reaction to treatment among the F0 males, the highest treatment level was reduced to 1.0% D911P after the sixth week of treatment.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily for clinical signs of treatment, and any animals found in distress were humanely sacrificed.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on a weekly basis, except for mated females, which were weighed on Days 0, 6, 13,
and 20 after mating, and Days 1, 4, 7, 14, and 21 of lactation.

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was monitored on a cage basis each week, except for mated females for whom food consumption was monitored on an individual basis on Days 0 through 5, 6 though 12, and 13 through 20 of gestation, and Days 1 through 6, 7 through 13, and 14 through 21 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

For the females, the duration and regularity of the oestrus cycle was established by daily vaginal smears, commencing 10 days before pairing.

Sperm parameters (parental animals):

An unmeasured sample of epididymal fluid was exuded by gentle squeezing, and the sperm allowed to diffuse into medium [M199 containing 0.5% bovine serum albumin (BSA)] at 37°C. A sample was taken for motility analysis of at least 200 sperm with the Hamilton Thorne IVOS Computer
Assisted Sperm Analyser (CASA). A sample of epididymal fluid (5 ml) was diluted with medium (995 ml), and an aliquot (50 ml) was set aside for
assessment of sperm morphology. The remainder was homogenised and stained with IDENT before counting by CASA, analysing 10 fields per animal. The aliquot taken for morphologic assessment was added to 4% neutral buffered formaldehyde (950 ml), applied to a microscope slide and stained with 1% nigrosin/eosin before assessment by light microscopy of at least 200 sperm. The frozen testis was thawed and then homogenised in SMT (0.9% saline, 0.01% merthiolate, 0.05% Triton X-100; 25 ml). An aliquot was stained with IDENT and counted as described previously.

Litter observations:

PARAMETERS EXAMINED
F0 offspring were examined approximately 24 h after birth (Postnatal Day [PND] 1) and the following were recorded for each litter: number of pups (live and dead), body weights of live offspring, sex ratio, and observations on individual pups. Litters of nine or more offspring were culled by random selection to eight, where possible four males and four females, on PND 4. At PND 25 males and females (one male and one female from each litter where possible) were randomly selected to produce the next (F1) generation.

The selected F1 females were examined for vaginal opening from Postnatal D (PND) 28, and the selected males were examined for preputial separation from PND 38. Examinations were made on a daily basis, and body weight was recorded on the day that vaginal opening or preputial separation was completed.

Postmortem examinations (parental animals):

Parental males were killed when the majority of litters had reached 2 weeks of age. Immediately after sacrifice, one epididymis (normally the left) was removed, weighed, and samples of fluid obtained from the cauda were diluted for assessment of sperm count, motility, and morphology. The
whole left testis was taken and frozen (220°C) before determination of homogenisation-resistant spermatid count.
Females were killed after their litters had been weaned. All animals were subject to complete gross necropsy, and any abnormal tissues were retained for further examination.
The following organs were also routinely removed: adrenal glands, brain, epididymides, kidneys, liver, caudal mammary gland, ovaries, oviduct, pituitary, prostate, seminal vesicles and coagulation gland, spleen, right testis, thymus, uterus and cervix, and vagina. Uteri were examined for the number of implantation sites [13]. Organs were stored in 4% neutral buffered formaldehyde, except the right testis, which was fixed in Bouin’s fluid for 24 h, before transfer into 70% industrial methylated spirit. In addition, liver sections from selected F1 male adults were frozen before analysis of cyanide-insensitive palmitoyl CoA oxidase activity.
HISTOPATHOLOGY
All organs from the control and high dose were examined microscopically, as well as those from the low and intermediate treatment groups that were found abnormal under macroscopic evaluation.

Postmortem examinations (offspring):

F0 offspring not selected for continuation, and all F1 offspring, were killed at PND 25 and examined macroscopically for abnormalities. Any abnormal tissues were retained for further examination. The following organs were also routinely removed: brain, epididymides, liver, ovaries, oviduct, prostate, seminal vesicles and coagulation gland, spleen, testes, thymus, uterus and cervix, and vagina. Offspring culled on PND 4 were not subjected to formal necropsy, unless grossly abnormal. All adults and weaned animals were killed by CO2 asphyxiation.
Offspring culled on PND 4, and any killed humanely before PND 10 were killed with pentobarbitone sodium, administered by intraperitoneal injection.
HISTOPATHOLOGY
All organs from the control and high dose were examined microscopically, as well as those from the low and intermediate treatment groups that were found abnormal under macroscopic evaluation.

Statistics:

For organ and body weight data, homogeneity of variance was assessed with Bartlett’s test. Where this was statistically significant, pair-wise comparison was performed with the Behrens–Fisher test, otherwise Dunnett’s test was used. Intergroup differences in macroscopic pathology and histopathology were assessed with Fisher’s exact test. Food-consumption and litter data were analysed with parametric tests (analysis of variance followed by Williams’ test) or nonparametric tests (Kru´skal–Wallis followed by Shirley’s test) as appropriate. For litter data, the litter was generally taken as being the unit for analysis. Because most distributions were non-normal, nonparametric analyses were routinely used.

Reproductive indices:

Pre-coital interval, gestation length, number of live litters, implantation sites, litter size

Offspring viability indices:

- Live birth index (live offspring on PND 1 compared to total litter size on PND 1)
- Viability index (live offspring on PND 4 compared to live offspring on PND 1)
- Lactation index (live offspring on day of examination [PND7 and PND21] compared to offspring on PND 4 after culling)

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significantly (p < 0.01) reduced growth in males of highest dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

significantly (p < 0.01) reduced growth in males of highest dose group

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

clear hepatotoxicity at the highest dose

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: reduced food intake in males of the highest dose group

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced gestation length in highest dose group

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No data shown

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Significantly reduced growth in F0 males within the first week of treatment, which accompanied reductions in food consumption and conversion efficiency. Male body weights in the highest dose groups were 75% to 80% of the concurrent control values after 6 weeks of exposure. The severity of this reaction was considered incompatible with the objectives of the study. Consequently, the highest level was reduced to 1.0% in the diet, for both males and females from week 7. From this point on, food conversion efficiency was the same in all groups, and body weight gain of males in the high exposure group was similar to control values. However, food consumption remained depressed in comparison to control, and body weights were consistently lower by about 20% to 25% in males until the time of mating. At the time mating commenced, mean body weights were 100%, 96%, and 76% of control in the 0.1%, 0.5%, and 1.0% D911P dose groups, respectively.
Females of both generations were less susceptible than the males. In the F0 generation, body weights of animals in the highest dose groups were only significantly affected by treatment with D911P, but were still within 10% of control values.
Overall, body weight gain by D911P exposed animals up to the time of mating was 86% of that in the control group.
Food consumption in female animals was similar for all treatment groups of both generations through gestation and lactation (not shown).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no apparent treatment-related effects on the regularity or duration of the oestrus cycle in all females of F0 and F1 generation

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Testicular spermatid count was significantly higher than control in all D911P-treatment groups of the F0 generation (p < 0.05). In the F1 generation no significant differences between treatment groups and controls occurred. Additionally, epididymal sperm counts and sperm quality were unaffected in F0 and F1 generation in all treatment groups. Therefore the increased testicular spermatid counts cannot be clearly attributed to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no apparent treatment-related effects on time to mating, fecundity or mating success in either generation.
Gestation length was slightly reduced in the highest dose group of F0 and F1 generations. The distribution of gestation lengths for animals exposed to 1.0% was significantly changed toward shorter duration compared to the control group. The nature of the shift in gestation length induced by D911P was to reduce the modal value by 0.5 days. Parturition, litter size, and offspring body weights were unaffected by treatment. There were no effects of treatment with D911P upon the number of implantation sites, litter size, or pup survival in either generation. Pup growth was largely unaffected, except for slightly reduced body weights of offspring in the highest dosage groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant changes in organ weights in both generations were generally limited to animals in the highest exposure group.
The weight of the seminal vesicles and prostate was reduced (p < 0.01) in highest dose group males. The weights of the testes were unaffected in all treatment groups. Animals exposed to D911P did not show significantly reduced ovary weight, but the weight of the uterus and cervix was reduced (p < 0.01) in the F0 generation. When organ weights were expressed in relation to body weight, most of the differences between the high-dose and other groups were no longer apparent. In the case of testes, epididymides (except F1 animals), and kidneys in males there was a significant increase in relative organ weight compared to control. However, uterus and cervix weight in the high dose group remained significantly depressed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic findings were largely limited to the livers of males in the high-dose groups. Livers were small, although lobes were swollen, pale, and showed areas of macroscopic change, whereas some had irregular surfaces. Surface changes in colour and texture were noted in a number of animals. There were low incidences of similar effects in males (swelling) and females (enlargement) of the intermediate- and high-dose groups.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The range of microscopic findings in the high-dose males was indicative of periacinar hepatotoxicity, with consequential increased cell turnover and regenerative hyperplasia.
Bile duct proliferation and congestion probably resulted from the altered architecture of the liver. Again, similar effects were noted at lower incidence in males of the intermediate dosage group, and very restricted effects (periacinar vacuolation and necrosis or hypertrophy) were noted in females. In addition, there was a significant increase in the activity of palmitoyl coenzyme A oxidase activity, a marker of peroxisomal proliferation, in the livers of F1 males in both the intermediate- and high-dose groups.
No other organs, including the reproductive organs, showed any macroscopic or histologic changes considered to be related to treatment.

OTHER INFORMATION:
The NOEL for liver in parental and F1 animals based on microscopic examination was found to be 60 mg/kg bw/d (0.1% in diet).

Dose descriptor:

NOAEL

Remarks:

reproductive health parameters and general systemic toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 0.5% in diet

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Remarks:

reproductive health parameters and general systemic toxicity

Effect level:

620 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 1.0% in diet

Sex:

male/female

Basis for effect level:

other: reduced parental body weight gain (F0 and F1 males), and reduced reproductive organ weights (males and females of F0 and F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weights of F1 male and female offspring in the highest exposure group during the last 2 weeks of lactation, reduced body weight gains in F1 males of the highest dose group

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

in F0 and F1 males and females of highest dose group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

changes in livers of the highest dose group

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

clear hepatotoxicity at the highest dose

VIABILITY (OFFSPRING)
No significant differences were found in live birth index, viability index and lactation index between treatment and control groups of F0 and F1 generations

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
After formal allocation of offspring to the F1 generation (approx. 28 days of age), body weights in all F1 males were comparable for the first 2 or 3 weeks of treatment after weaning. However, from about the fifth week, males in the highest dose group exhibited reduced body weight gain compared to controls. As in the case of the F0 generation, this response accompanied slight reductions in food consumption and conversion. The deviation from control values of body weights in the highest dose groups increased from 4 to 6 weeks, reaching a nadir of about 90% by week 6.
Females of both generations were less susceptible than the males. In the F0 generation, body weights of animals in the highest dose groups were only significantly affected by treatment, but were still within 10% of control values.
Females of the F1 generation were unaffected by treatment, and food consumption and conversion efficiency were similar for all groups of animals.
Mean pup weights of F1 male and female offspring (F2) were reduced in the highest exposure group during the last 2 weeks of lactation.

SEXUAL MATURATION (OFFSPRING)
The age of onset of sexual maturation—defined as the day at which preputial separation and vaginal opening were completed—in females was similar in all groups. Preputial separation in males appeared to be delayed by ~1 day in the highest dose group, but this apparent difference was not statistically significant (p < 0.05) and the values were within the range of laboratory historical controls.

ORGAN WEIGHTS (OFFSPRING)
Statistically significant changes in organ weights in both generations were generally limited to animals in the highest exposure group.
The weight of the seminal vesicles and prostate was reduced (p < 0.01) in highest dose group males. The weights of the testes were unaffected in all treatment groups. Animals exposed to D911P did not show significantly reduced ovary weight, but the weight of the uterus and cervix was reduced (p < 0.01) in the F0 generation. When organ weights were expressed in relation to body weight, most of the differences between the high-dose and other groups were no longer apparent. In the case of testes, epididymides (except F1 animals), and kidneys in males there was a significant increase in relative organ weight compared to control. However, uterus and cervix weight in the high dose group remained significantly depressed.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic findings were largely limited to the livers of males in the high-dose groups. Livers were small, although lobes were swollen, pale, and showed areas of macroscopic change, whereas some had irregular surfaces. Surface changes in colour and texture were noted in a number of animals. There were low incidences of similar effects in males (swelling) and females (enlargement) of the intermediate- and high-dose groups.
There were no macroscopic changes in offspring killed before weaning or at 25 days of age that were attributed to treatment (not shown);

HISTOPATHOLOGY (OFFSPRING)
The range of microscopic findings in the high-dose males was indicative of periacinar hepatotoxicity, with consequential increased cell turnover and regenerative hyperplasia.
Bile duct proliferation and congestion probably resulted from the altered architecture of the liver. Again, similar effects were noted at lower incidence in males of the intermediate dosage group, and very restricted effects (periacinar vacuolation and necrosis or hypertrophy) were noted in females. In addition, there was a significant increase in the activity of palmitoyl coenzyme A oxidase activity, a marker of peroxisomal proliferation, in the livers of F1 males in both the intermediate- and high-dose groups.
No other organs, including the reproductive organs, showed any macroscopic or histologic changes considered to be related to treatment.

Dose descriptor:

NOAEL

Remarks:

reproductive health parameters and general systemic toxicity

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 0.5% in diet

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOAEL

Remarks:

reproductive health parameters and general systemic toxicity

Generation:

F1

Effect level:

620 mg/kg bw/day

Based on:

test mat.

Remarks:

equivalent to 1.0% in diet

Sex:

male/female

Basis for effect level:

other: reduced parental body weight gain (F0 and F1 males), transient depression of offspring (F2) body weight gain (males and females) and reduced reproductive organ weights (males and females of F0 and F1)

Reproductive effects observed:

not specified

Completion of preputial separation (PS) and vaginal opening (VO) in animals of the F1 generation exposed to D911P in the diet

	Dietary Concentration (%) D911P
	0	0.1	0.5	1.0	Historical Control^a
Males  Age at PS (d)	44.8±2.3 (41-50)	44.6±2.5 (41-50)	44.6±2.7 (41-50)	46.1±2.8 (43-52)	43.7-47.3^b 41-57^c
Bodyweight at PS (g)	235±22	229±27	228±23	237±26
Females  Age at VO (d)	34.7±2.0 (30-38)	35.3±2.3 (32-41)	34.9±3.3 (30-42)	35.0±2.2 (31-41)	33.0-35.7^b 29-40^d
Bodyweight at VO (g)	128±16	123±17	119±18	122±16

^alaboratory historical control ranges for CD rats

^bgroup mean values, n = 7

^cindividual animal values, n = 197

^dindividual animal values, n = 198

Testicular and epididymal sperm counts and sperm quality in F0 and F1 rats exposed to D911P in the diet

	Dietary D911P Concentration (%)
	F^0Generation				F^1Generation
	0	0.1	0.5	2.0/1.0^a	0	0.1	0.5	1.0
Number of animals	27	28	28	28	28	27	28	27
Testis spermatid count (10^6/g)	108±33	139±49*	134±41*	136±51*	122±43	123±45	114±37	115±50
Epididymis sperm evaluation  Sperm count (10^7/mL)  Motile (%)  Progressively motile (%)  Normal morphology (%)	219±47 76±8 40±9   96±3	227±71  75±10 40±10   95±4	223±58 73±11 38±9   95±3	206±61 73±15 39±10   93±11	215±60 77±8 38±7   91±9	200±55 77±8 38±8   92±7	199±36 77±6 40±7   89±11	201±50 75±8 37±7   89±12

^a2.0% from week 1 through to week 3, 1.0% thereafter

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

388 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for grouping of substances and read-across

There are only limited data available on the toxicity to reproduction of 1,2-Benzenedicarboxylic acid, di-C16-C18-alkyl esters (CAS 90193-76-3). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of reproduction toxicity

CAS	Toxicity to reproduction
90193-76-3 (a)	RA: CAS 68515-43-5
68515-43-5 (b)	2 -generation study: NOAEL = 300 mg/kg bw/day

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substance is considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 1,2-Benzenedicarboxylic acid, di-C16-C18-alkyl esters (CAS 90193-76-3). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion

CAS 68515-43-5

The toxicity to reproduction of 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (CAS No. 68515-43-5) has been investigated in a two generation toxicity study according to EPA OPPTS 870.3800.

A two generation oral feeding study on reproduction and fertility effects was performed with 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (CAS No. 68515-43-5) according to EPA OPPTS 870.3800 (Willoughby, 2000). The major treatment-related effect was a severe reduction in adult male body weight gain, with a lesser effect in females. There was no treatment-related deterioration of fertility or fecundity at any dose level. The only effect upon reproductive parameters attributable to treatment was a reduction in epididymides weight at the highest dose level of 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters. However, in the absence of functional or histopathologic changes, the significance of these observations is unclear.

Transiently reduced body weight of offspring in both generations may be a secondary response to depression of maternal body weight gain or a direct effect of the test substance in the diet resulting from pups eating it. Reductions in organ weights (except liver) are also attributed to a nonspecific response to reduced body weight. The livers of male rats, and to a lesser extent females, showed the classic biochemical, morphologic, and histologic changes that would be expected of a substance of the peroxisome proliferator class—specifically hypertrophy, hyperplasia, and an increase in the activity of palmitoyl CoA oxidase.

The NOAEL for effects upon reproductive health parameters and general systemic toxicity for 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters was 0.5% in the diet (approx. 300 mg/kg bw/day) for F0 and F1 animals. This NOAEL was based on transient depression of offspring body weight gain (males and females), reduced parental body weight gain (males), and reduced reproductive organ weights (males and females).

 

Additionally, a 90-d oral feeding study with 1,2-Benzenedicarboxylic acid, mixed cetyl and stearyl esters (CAS No. 68442-70-6) was performed similar to OECD guideline 408 (Rivett, 1972). Upon oral feeding with doses up to 1200 mg/kg bw/day no histopathologic changes were observed in ovaries and testes.

 

Conclusion for toxicity to reproduction

A two generation study is available for 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (CAS No. 68515-43-5) with a NOAEL of 300 mg/kg bw/day based on adverse effects on transient depression of offspring body weight gain (males and females), reduced parental body weight gain (males), and reduced reproductive organ weights (males and females). In a 90-d oral feeding study with doses up to 1200 mg/kg bw/day no histopathologic changes were observed in ovaries and testes.

Short description of key information:
A NOAEL for fertility of 300 mg/kg bw/d in parental and F1 rats was determined in a two-generation reproduction toxicity study with the Read Across substance 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (MW 418.62 – 474.73). This would refer to an equimolar NAEL of 388 mg/kg bw/d for 1,2-Benzenedicarboxylic acid, di-C16-18-alkyl esters (MW 614.98 – 671.09) using a conversion factor of 1.29 (614.98 / 474.73).

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from structural analogues. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substances and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information

The equimolar NAEL of 1290 mg/kg bw/d for developmental toxicity for 1,2-Benzenedicarboxylic acid, di-C16-18-alkyl esters (MW 614.98 – 671.09) was derived from the developmental NOAEL value established at 1000 mg/kg/day for the Read Across substance 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (CAS No. 68515-43-5, MW 418.62 – 474.73) using a conversion factor of 1.29 (614.98/474.73).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study with acceptable restrictions. Not full study report published; only data of C9-C11 alkyl phthalates were presented here;

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Groups of 22 timed-mated Sprague-Dawley rats were administered 250, 500, or 1000 mg/kg D911P daily by oral gavage (5 mL/kg) between gestation days (GD) 1 and 19. Control animals received the vehicle (olive oil) alone. On GD20, the animals were sacrificed and the fetuses examined.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd. (Margate, Kent).
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 210-267 g
- Diet: ad libitum SDS Laboratory Animal Diet No. 1, Special Diet Services Ltd, Witham, Essex
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%):55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Dose volume: 5 mL/kg bw

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm-positive vaginal smear or at least three copulation plugs referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestational days 1-19

Frequency of treatment:

once a day

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0, 250, 500, and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweight was measured daily throughout the study, and

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
food consumption was recorded for gestation days 1–2, 3–5, 6–8, 9–11, 12–14, 15–17, and 18–19.

POST-MORTEM EXAMINATIONS: Yes
On GD20, the females were killed by carbon dioxide inhalation, weighed, and examined macroscopically for signs of disease or adverse reaction to treatment.

Ovaries and uterine content:

The reproductive tracts, including ovaries, were removed and assessed for gravid uterine weight, number of corpora lutea in each ovary, numbers of implantation and resorption sites, and number and distribution of fetuses in each uterine horn.

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Fetuses were uniquely identified with respect to their position in the uterine horn. Each fetus was weighed, sexed, examined for external abnormalities, and euthanized by chilling on a cool plate at approximately 4°C. Each placenta was weighed and examined for abnormalities. Approximately half of each litter was allocated to skeletal examination. These fetuses were given a visceral examination prior to evisceration and fixation in industrial methylated spirit. They were then processed and stained with Alizarin Red, using a modification of the Dawson staining technique. The remainder of the fetuses were fixed in Bouin’s fluid prior to examination by the Wilson free-hand serial sectioning technique.

Statistics:

Homogeneity of variance was assessed using Bartlett’s test, and data were analyzed with parametric tests (analysis of variance followed by Williams’ test) or nonparametric tests (Kru´skal-Wallis followed by Shirley’s test) as appropriate. Data on fetal malformations and variations was analyzed using categorical data models. Significant differences between treatments and controls for the proportions of affected fetuses were determined using Wald x2 tests with Bonferroni’s adjustment for multiple testing applications. Trend analysis was performed using logistic regression to separate spurious results from the statistically significant effects of an increased dosage level. For litter data, the litter was generally taken as being the unit for analysis. Since most distributions were nonnormal, nonparametric analysis was routinely used.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Treatment resulted in no signs of maternal toxicity, as assessed by adjusted maternal bodyweight gain throughout gestation, clinical examinations and food consumption. There were no remarkable macroscopic findings in the maternal animals at necropsy.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Treatment resulted in no effects upon litter size, fetal survival or bodyweight. Fetal, litter, and placental weights were all unaffected by treatment with D911P, and there was no effect upon the overall incidence of external or visceral abnormalities.
There was a significant increase in dilated renal pelves in pups of the high dose groups.
Increased incidences of rudimentary supernumerary lumbar ribs at 500 and 1000 mg/kg bw /d.
These rudimentary supernumerary ribs, defined as being less than 50% the length of the 13th rib, are a common skeletal variation that occurs spontaneously at a high frequency in rats.

Remarks on result:

other: not specified

Abnormalities:

not specified

Developmental effects observed:

not specified

 Table 1

Selected foetal observations following treatment with D911P from GD1 to GD19

	0	250	500	1000	Historical Control^d
No. examined viscerally^a	165 (22)	162 (22)	157 (22)	153 (21)
No. examined ekeletally^a	168 (22)	158 (22)	158 (22)	157 (2!)
Visceral findings^b
dilated ureter	3 (3) [1.8]	11 (5) [8.6]	4 (3) [2.4]	11 (7) [7.7]	1.9-1.3% (13.6-45.5%)
dilated renal pelvis^c	2 (2) [1.0]	7 (3) [4.1]	2 (1) [1.3]	12 (5) [7.9*]^‡
hydroureter^c	0 [0.0]	10 (6) [6.9]	1 [0.6]	8 [3] [5.3]
Skeletal findings^b
lumbar ribs (13/14 or 14/14)	23 (13) [13.6]	24 (10) [15.9]	45 (17) [28.2**]	40 (14) [26.7*]^‡	8.5-16.5% (33.3-72.7%)
cervical ribs	0 [0.0]	2 (2) [1.2]	4 (3) [2.5]	0 [0.0]	0.0-2.0% (0.4-4.8%)
complete 14^thribs	0	0	2 (1)	1

^aData represent the number foetuses (litters) examined in each group.

^bData represent the number foetuses (litters) affected in each group, and [foetus affected per litter (%)].

^cData from foetuses processed for skeletal examination.

^dRange of control incidences from 9 studies in Sprague-Dawley rats conducted at the same laboratory over the preceding 18 months. Control animals received olive oil (5 studies), water (1 study), Tween 80 (1 study), or 1% aqueous methylcellulose (2 studies).

* p < 0.05 compared to control

** p < 0.01 compared to control

^‡Significance for trend, p < 0.01

Conclusions:

In conclusion, D911P has no potential to adversely affect development in the rat. Administration of doses up to 1000 mg/kg/day throughout gestation induced no detectable toxicity in the dams, and resulted only in minor skeletal and visceral variations (non-adverse). There was no evidence of malformations following treatment. The NOAEL for maternal and developmental toxicity is therefore established at 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substance.

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

Developmental toxicity/teratogenicity

Justification for grouping of substances and read-across

There are only limited data available on the developmental toxicity of 1,2-Benzenedicarboxylic acid, di-C16-C18-alkyl esters (CAS 90193-76-3). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of reproduction toxicity

CAS	Developmental toxicity
90193-76-3 (a)	RA: CAS 68515-43-5 RA: CAS 68442-70-6
68515-43-5 (b)	NOAEL = 1000 mg/kg bw/day
68442-70-6	NOAEL ≥ 90 mg/kg bw/day

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substance is considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 1,2-Benzenedicarboxylic acid, di-C16-C18-alkyl esters (CAS 90193-76-3). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

CAS 68515-43-5

A prenatal developmental toxicity study was performed with the read across substance 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (D911P, CAS No. 68515-43-5) according to EPA OPPTS 870.3700 (Fulcher, 2001). Groups of 22 timed-mated Sprague-Dawley rats were administered 250, 500, or 1000 mg/kg D911P daily by oral gavage (5 mL/kg) between gestation days (GD) 1 and 19. Control animals received the vehicle (olive oil) alone. On GD20, the animals were sacrificed and the fetuses examined. In this study the only findings in fetuses were increased incidence of rudimentary supernumerary ribs at 500 mg/kg bw/d and increased incidence of dilated renal pelves at 1000 mg/kg bw/d. Rudimentary supernumerary ribs, defined as being less than 50% the length of the 13th rib, are a common skeletal variation that occurs spontaneously at a high frequency in rats. This variation is spontaneously reversible in rats and its incidence diminishes as the animals mature. In conclusion, D911P has a low potential to adversely affect development in the rat. Administration of doses up to 1000 mg/kg/day throughout gestation induced no detectable toxicity in the dams, and resulted only in minor skeletal and visceral variations (non-adverse). There was no evidence of malformations following treatment. The NOAEL for maternal and developmental toxicity is therefore established at 1000 mg/kg/day.

 

CAS 68442-70-6

1,2-Benzenedicarboxylic acid, mixed cetyl and stearyl esters (CAS No. 68442-70-6) was tested for its teratogenicity in a Prenatal Developmental Toxicity Study performed equivalent to OECD Guideline 414 (Derache, 1983). The highest tested dose of only 90 mg/kg bw/day did not induce abnormalities in the implantation rates, number of abortions, number of resorptions or number of living fetuses. No malformations or nanisms occurred in the control and treated groups. Litter sizes and average body weight of fetuses were comparable between control and treated animals.

 

Conclusion for developmental toxicity/teratogenicity

Two studies investigating the developmental toxicity via the oral route are available for the structural analogues 1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters (D911P, CAS No. 68515-43-5) and 1,2-Benzenedicarboxylic acid, mixed cetyl and stearyl esters (CAS No. 68442-70-6). Due to no observed adverse effetcs the NOAEL for maternal and developmental toxicity was set at 1000 mg/kg bw/day.

Effects on developmental toxicity:

A waiver for the requirement to perform a prenatal developmental toxicity study in a 2nd species was included, as this requirement is considered not to add new information for hazard assessment and therefore is scientifically and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified.

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from structural analogues. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substances and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on read-across from the structurally similar substances, the available data on the toxicity to reproduction do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.