Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (according to OECD 421), rat: NOAEL(systemic) = 300 mg/kg bw/day (m/f); NOAEL (reproduction toxicity) ≥1000 mg/kg bw/day (m/f) (RA from source substance Dibutyl adipate (CAS 105-99-7))

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 8 wks;
- Weight at study initiation: males: 270.6 - 326.5 g; females: 199.5 - 243.4 g
- Fasting period before study: No
- Diet: CA-1 by CLEA Japan, Meguro, Japan, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle: due to the lipophilic properties of the test material, corn oil was selected as the appropriate vehicle
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Detailed method was not described. The measured concentration was between 98.3 and 104% of nominal concentration.

Duration of treatment / exposure:

Males: 14 days before mating and for 28 days thereafter
Females: total of 42-53 days beginning 14 days before mating to day 3 of lactation

Frequency of treatment:

7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A dose finding study was performed. Test solutions of 0, 500 and 1000 mg/kg bw/day were administered for 14 days to female rats. Slight salivation was observed during treatment but no mortality occured. No difference in body weight and food consumption was observed and necropsy revealed no abnormalities.No change in organ weight compared with control group was observed.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, before and after administration

BODY WEIGHT: Yes
- Time schedule for examinations: Males on Days 1, 8, 15, 22, 29, 36 and 43, females on Days 1, 8 and 15 of treatment, Days 0, 7, 14 and 20 of gestation and Days 0 and 4 of lactation

FOOD CONSUMPTION: not examined

Oestrous cyclicity (parental animals):

Observed

Sperm parameters (parental animals):

Parameters examined in parental animals:
testis weight, epididymis weight, other: histopathology

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 43
- Maternal animals: All surviving animals on Day 4 of lacation

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weight: kidneys and spleen of both sexes and testes and epididymides of males.
Microscopic observation: testes and epididymides of males; ovary and uterus of females

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

Bartlett's test, Dunett's test, Scheffe's test, Kruskal-Wallis test

Reproductive indices:

copulation index (no. of copulated rats/no. of mated rats x 100),
fertility index (no. of pregnant females/no. of females with successful copulation x 100),
gestation index (no. of females with live pups/no. of pregnant females x 100),
implantation index (no. of implantation sites/no. of corpora lutea x 100),
delivery index (no. of pups born/no. of implantation sited x 100)

Offspring viability indices:

birth index (no. of live pups on day 0/no. of implantation sites x 100),
live birth index (no. of live pups on Day 0/no. of pups born x 100),
viability index (no. of pups alive on Day 4 of lactation/no. of pups born x 100)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent increase in number of rats with salivation after dose administration in both sexes.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Slight salivation was observed in treatment group with dose-responsibility (see Table 1). This change was not regarded as neurological effect but caused by stimulation by the administration of the test compound.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Light suppression of body weight gain was observed in males in 1000 mg/kg bw/day group but there was no significant difference.

ORGAN WEIGHTS (PARENTAL ANIMALS)
An increase in relative weight of kidney was observed in both sexes of 1000 mg/kg bw/day group. An increase in relative weight of spleen in males of 100 and 1000 mg/kg bw/day, increase in absolute weight of spleen in females of 100 mg/kg was observed ( see Table 2).

HISTOPATHOLOGY (PARENTAL ANIMALS)
Infiltration of lymphocyte in testis was observed in control and 1000 mg/kg bw/day group. This change was not related to the test compound.

OTHER FINDINGS (PARENTAL ANIMALS)
A dam in 300 mg/kg bw/day group didn't retrieve pups and all pups were dead until on PND 2. But this change was not regarded as compound-related effect.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: organ weights: kidney weights increased

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for reproductive parameters is corresponding to the highest dose tested

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Pup viability on PND 4 was decreased.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: pup weight on PND 0 and 4 was slightly decreased (without reaching statistical significance)

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
1000 mg/kg bw/day: Pup viability on PND 4 was statistically significantly decreased (see Table 3).

CLINICAL SIGNS (OFFSPRING)
No effects were observed.

BODY WEIGHT (OFFSPRING)
1000 mg/kg bw/day: pup weight on PND 0 and 4 was slightly decreased (without reaching statistical significance (see Table 3).

GROSS PATHOLOGY (OFFSPRING)
No effects were observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pup weight; viability index

Key result

Reproductive effects observed:

not specified

Table 1: Number of animals showing salivation upon treatment [male/female]

	Dose [mg/kg bw/day]
Days of treatment	0	100	300	1000
1-7	-	-	-	12/9
18-14	-	-/1	4/4	13/13
15-21	-	3/2	7/9	13/13
22-28	-	-	2/1	12/13
29-35	-	-	6/4	13/13
36-42	-	-	6/3	13/12
43	-	-	-	-
Total	-	3/3	11/9	13/13

 

Table 2: Organ weights

Males after 42 days of treatment		Dose [mg/kg bw/day]
Kidney		0	100	300	1000
absolute weight [g]	mean	2.96	3.12	3.04	3.08
	SD	0.39	0.34	0.32	0.35
relative weight [% of mean body weight]	mean	0.6	0.61	0.63	0.65*
	SD	0.05	0.04	0.06	0.06
Spleen
absolute weight [g]	mean	0.79	0.95	0.84	0.9
	SD	0.14	0.19	0.11	0.2
relative weight [% of mean body weight]	mean	0.16	0.18*	0.17	0.19**
	SD	0.02	0.03	0.02	0.03
Females at Day 4 of lactation
Kidney
absolute weight [g]	mean	1.95	1.97	2.1	2.11
	SD	0.15	0.19	0.22	0.17
relative weight [% of mean body weight]	mean	0.6	0.58	0.62	0.65
	SD	0.04	0.03	0.05	0.07
Spleen
absolute weight (in g)	mean	0.59	0.74*	0.65	0.68
	SD	0.08	0.16	0.13	0.09
relative weight (% of mean body weight)	mean	0.18	0.22	0.19	0.21
	SD	0.03	0.04	0.03	0.03

 

Table 3: Reproductive parameters

Dose [mg/kg bw/day]		0	100	300	1000
No.of female animals:		13	13	13	13
No. of mated pairs:		13	13	13	13
No. of copulated pairs:		13	13	13	13
Copulation index [%]		100	100	100	100
No. of pregnant females		12	12	12	12
Fertility index [%]		92.3	92.3	92.3	92.3
Pairing day until copulation
	mean	4.1	2.6	3	2.7
	SD	3.3	0.8	2.3	1
Frequency of vaginal estrus
	mean	1.1	1	1.2	1
	SD	0.3	0	0.6	0
No. of pregnant females with pups alive:		12	12	12	12
Gestation index (%)		100	100	100	100
Gestation length (days)
	mean	22.3	22.1	22.2	22.2
	SD	0.5	0.3	0.4	0.4
No. of corpora lutea
	mean	15.7	15.6	17	16.9
	SD	2	1.6	1.9	1.7
No.of implantation site
	mean	14.8	14.7	15.8	15.5
	SD	2.6	2.1	3.1	2.1
Implantation index [%]
	mean	94.2	94	92.2	91.5
	SD	7.8	8.4	14.1	7.5
Day 0 of lactation
No.of pups born
	mean	13.6	14.2	15	14.6
	SD	2.4	1.9	2.9	2.3
Delivery index [%]
	mean	91.8	96.7	95.5	93.9
	SD	6.5	3.4	5.7	5.9
No.of pups alive
	mean	13.3	13.9	14.3	13.7
	SD	2.4	1.9	2.6	3.3
Birth index (%)
	mean	90.1	95.2	91.8	88.2
	SD	6.1	6	8.8	16.8
Live birth index (%)
	mean	98.2	98.4	96.2	93.6
	SD	4.6	4.1	8.4	15.2
Pups weight [g]
Male	mean	6.5	6.4	6.2	6.1
	SD	0.7	0.6	0.8	0.7
Female	mean	6.1	6	5.8	5.8
	SD	0.6	0.6	0.7	0.5
Sex ratio on day 0 of lactation [no. of male pups/total no. of pups]
	mean	47.6	41.4	49.4	48.6
	SD	13	7.7	12.7	11.9
Day 4 of lactation
No.of pups alive
	mean	13.3	13.8	13.1	13.3
	SD	2.4	1.7	4.8	3.5
Viability index [%]
	mean	100	99.5	90.6	96.1*
	SD	0	1.7	28.6	5.3
Pups weight [g] on day 4 of lactation
Male	mean	10.6	10.8	10.2	9.9
	SD	1.8	1.2	1.7	1.9
Female	mean	10.1	10.1	9.7	9.3
	SD	1.7	1.2	1.6	1.4

 SD standard deviation

* significant difference from control p <0.05

** significant difference from control p <0.01

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: organ weights: kidney weights increased

Remarks on result:

other: source: CAS 105-99-7

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for reproductive parameters is corresponding to the highest dose tested

Remarks on result:

other: source: CAS 105-99-7

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: pup weight; viability index

Remarks on result:

other: source: CAS 105-99-7

Key result

Reproductive effects observed:

not specified

Conclusions:

In an oral reproduction/developmental toxicity screening test with the source substance Dibutyl adipate (CAS 105-99-7) the NOAEL for reproductive toxicity in parental animals was established at ≥ 1000 mg/kg bw/day, while the NOAEL for systemic toxicity in parental animals was set at 300 mg/kg bw/day. As explained in the analogue justification, these results are considered to be valid also for the target substance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study from an analogue source substance. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to analogue justification for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

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 read-across

There are no experimental data on reproduction toxicity available for Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3). To fulfil the standard data requirements defined in Regulation (EC) No. 1907/2006, Annex VIII, 8.7 read-across from appropriate substances is conducted in accordance with Regulation (EC) No. 1907/2006, Annex XI.

According to 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) “to avoid the need to test every substance for every endpoint”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read- across approach is provided in the technical dossier (see IUCLID Section 13).

As no experimental data are available on reproduction toxicity, read-across of reliable data on the analogue substance Dibutyl adipate (CAS 105-99-7) was conducted.

CAS 105-99-7

An oral reproduction/developmental toxicity screening test with Dibutyl adipate (CAS 105-99-7) was performed in Sprague-Dawley rats according to OECD 421 and in compliance with GLP (MHLW, 1996, WoE). Dilutions of the test substance in corn oil were administered once daily to groups of 13 animals per sex at dose levels of 100, 300 and 1000 mg/kg bw/day via oral gavage. A control group treated according to the same protocol received the vehicle only. Females were exposed for a total period of 42-53 days, including 14 days before mating and until Day 3 of lactation, whereas males were treated 14 days before mating and 28 days thereafter. After administration, clinical signs involved a dose-dependent increase in salivation in animals of both sexes. However, the increase in salivation was not regarded as neurological effect, but caused by stimulation by the administration of the test substance. A slight, but non-significant suppression of body weight gain was observed in males at 1000 mg/kg bw/day. The relative organ weight of kidney was statistically significantly increased in both sexes at 1000 mg/kg bw/day. In addition, an increase in the relative weight of spleen in males treated with 100 and 1000 mg/kg bw/day was observed, while the only change in spleen weight noted in females involved the increase in absolute weight at 100 mg/kg bw/day. No effects on reproductive function (sperm parameters and oestrus cyclicity) and performance (copulation, fertility, gestation, implantation and delivery index) were observed after treatment compared to controls in any of the parental animals. Testis weight, epididymis weight, and histology of these organs did not reveal any substance-related effects in males. Effects on offspring included a slight, although statistically significant decrease (96.1%) in the pup viability on Day 4 of lactation at the 1000 mg/kg bw/day dose group compared with controls (100%). At the same dose, a slight decrease in pup weight was observed on Day 0 and Day 4 of lactation, without reaching statistical significance. Based on the results of this study, the NOAEL for reproductive toxicity was established at ≥ 1000 mg/kg bw/day, while the NOAEL for systemic toxicity in parental animals was set at 300 mg/kg bw/day.

Conclusion for toxicity to reproduction

A reproduction/developmental toxicity screening test with the source substance Dibutyl adipate (CAS 105-99-7) showed that no adverse effects on fertility via the oral route up to the limit dose recommended in the relevant guideline. Thus, as the available data did not identify any hazard for toxicity to reproduction, Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) is not expected to be hazardous following oral exposure.

Effects on developmental toxicity

Description of key information

Oral (according to OECD 421), rat: NOAEL(systemic) = 300 mg/kg bw/day (m/f); NOAEL (F1) = 300 mg/kg bw/day (m/f) (RA from source substance Dibutyl adipate (CAS 105-99-7))

Dermal (similar to OECD 414; focus: heart development), rat: NOAEL (maternal toxicity) ≥ 2000 mg/kg bw/day; NOAEL (developmental toxicity) ≥ 2000 mg/kg bw/day (no confirmation of levocardia; RA from source substance Ditridecyl adipate (CAS 16958-92-2))

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study report, comparable to OECD guideline study with acceptable restrictions (non-GLP, no data on analytical purity)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(adopted 2001)

Deviations:

yes

Remarks:

15 females/group instead of 20 females/group; only two dose levels tested instead of three as recommended by guideline, no GLP, no data on test item purity)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: control: 237.2 ± 7.3 g; low dose: 239.8 ± 8.7 g; high dose: 234.9 ± 10.1 g
- Fasting period before study: no
- Housing: individually
- Diet: Purina Certified Rodent Chow #5002 (Meal); ad libitum
- Water: tap water, via an automatic watering system; ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: clipped, intact dorsal skin
- Type of wrap: open application, no wrap used
- Time intervals for shavings or clipplings: on gestation day 0 and once weekly thereafter

REMOVAL OF TEST SUBSTANCE
- Washing: no washing

TEST MATERIAL
- Constant volume or concentration used: yes
- The test material was measured using a 1.00 mL syringe (calibrated in 0.01 mL) for both groups, and during dispensing was spread evenly on the clipped dorsal skin of the rats using the tip of the syringe. (No needle was used.)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
To minimize ingestion of the test material, the rats were fitted with cardboard Elisabethan-style collars. These collars were lined with latex tubing to minimize the development of irritation or lesions.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- If cohoused: cohoused with male rats from in-house breeder population
- M/F ratio per cage: 1:1
- Duration of mating period: 5 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation days 0-19

Frequency of treatment:

once daily

Duration of test:

20 days

Dose / conc.:

800 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of a 13-week study conducted previously with the same material (ExxonMobil, Project 40552).
- Dermal control: Presumed-pregnant rats were clipped as the animals from the treatment groups. The intact dorsal skin of each rat was stroked with the tip of a 1.00 mL syringe, but no test material was applied.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: mortality, clinical signs of ill-health (pathosis), appearance, abortion, premature delivery, behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6, 10, 13, 16, and 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal was calculated for gestation day intervals 0-3, 3-6, 10-13, 13-16, and 16-20.

CLINICAL CHEMISTRY: Yes
- Blood samples were collected at the time of sacrifice from the abdominal aorta of each rat and allowed to clot in an SST Serum Separator Tube (Beckman-Dickinson, Rutherford, NJ).
- Parameters:
- Alanine aminotransferase (ALT),
- albumin,
- albumin/globulin ratio,
- alkaline phosphatase (ALP),
- aspartate aminotransferase (AST),
- total bilirubin,
- calcium,
- chloride,
- cholesterol,
- creatine,
- globulin,
- glucose,
- iron,
- lactate dehydrogenase (LDH),
- inorganic phosphorus,
- potassium,
- sodium,
- sorbitol dehydrogenase (SDH),
- total protein,
- triglycerides,
- urea nitrogen,
- uric acid.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: The thoracic and abdominal cavities were opened and all organs were examined grossly for evidence of pathological changes.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Parameters: gender, body weight, length, gross external examination

- Soft tissue examinations: Yes: half per litter
- Fetuses were fixed in Bouin's solution, stored in 70% Ethanol and evaluated. Visceral analysis was performed usng a modification of the Wilson technique of free-hand sectioning by razor blade.

- Skeletal examinations: Yes: half per litter
- Fetuses were peeled, eviscerated, fixed in 95% ethanol, macerated in potassium hydroxide, differentially stained for cartilage and bone, cleared in glycerine, and examined for skeletal anomalies.

- Head examinations: Yes: half per litter

Statistics:

Maternal biophase and caesarean section data, and fetal data were evaluated by analysis of variance followed by group comparisons using Fisher's Exact or Dunnet's Test. Fetal skeletal data were evaluated statistically by ANOVA followed by group comparisons using Fisher's Exact Test. Fetal visceral data were evaluated statistically using Fisher's Exact Test. Statistical analyses of clinical chemistry data were performed separately on individual serum components using SAS procedures. First the F-test was employed to do an analysis of variance on the serum data obtained from control and exposed groups. Next, the Student-Newman-Keul's multiple comparison test was employed to identified the specific group subsets within the serum data sets identified as having nonrandom variance. The observed statistically significant differences were reported only when a dose-related effect was found in the treated animals.
In general, for all statistical tests, differences between control and treated groups were considered statistically significant if the probability of the difference being due to chance was less than 5% (p<0.05).

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Body weights were statistically significantly decreased in the animals of the high-dose group. Statistically significant differences between the serum data from control and treated rats were observed for a number of parameters.

Details on maternal toxic effects:
- Observations during gestation:
Red nasal exudate, chromodacryorrhea and neck lesions were observed equally in control and treated animals. According to the author of the study, these are common findings in animals that were collared.
Signs of dermal irritation, including erythema and flaking of the skin, were observed in most of the animals from both treatment groups. In general, the irritation was very mild. Scabs were observed at the side of test substance application in two animals exposed to 800 mg/kg bw/day.

- Maternal body weights and food consumption:
In general, the mean body weight of the pregnant rats from all of the groups increased throughout gestation, following what would be regarded as a normal growth curve of weight gain. However, animals from the high-dose group gained significantly less (p<0.01) weight during the first and last interval of gestation, which subsequently contributed to the reduced weight gain throughout the entire period of gestation. Animals from the low-dose group demonstrated a significant reduction in body weight gain only during gestation days 0-3. Overall net body weight change was significantly lower in the 2000 mg/kg bw/day-group when compared to the control. With the exception of the first two intervals of food consuption recording, animals exposed to the substance consumed more food than control animals. It should be noted, however, that the total amount of food consumed during gestation by each of the experimental groups was comparable.

- Observations of dams at caesarean section:
At the time od necropsy, no findings attributable to exposure to the test substance were observed.

- Clinical chemistry analyses:
Statistically significant differences between the serum data from control and treated rats were observed for glucose, ALT, alkaline phosphatase, creatinine, cholesterol, triglycerides, total protein, iron, and globulin. A linear relationship (>99% confidence level) was found between dose and serum level for these components except glucose and ALT. When the historical serum reference values were taken into consideration, a portion of the dose-response curves for alkaline phosphatase, cholesterol, total protein, and globulin fall outside the normal range.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: The decrease in body weight seen at 2000 mg/kg bw/d was not considered to be an adverse effect.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: In the fetuses exposed to the test substance at 2000 mg/kg bw/day a statistically significantly increased incidence of levocardia was observed. In addition, in the high-dose fetuses an increased incidence of hydronephrosis was noted.

Details on embryotoxic / teratogenic effects:
- Fetal body measurements:
Mean fetal body weights and crown-rump lengths were not affected by exposure to the test substance.

- Fetal examinations:
With the exception of one fetus exposed in utero to the test substance at 2000 mg/kg bw/day, no remarkable findings were observed during external examination of the fetuses. The mentioned fetus, in addition to being edematous, had a shortened thoracic-lumbar (trunk) region, malrotated hindlimbs, absence of digits (ectrodactyly) on its forepaws, and no tail (acaudia). Due to their occurrence in only one fetus (fetal incidence = 1/191 = 0.5%), these anomalies were probably not related to test substance exposure.
A variety of fetal skeletal anomalies were observed in all groups at a low incidence. They were probably not related to substance exposure due to their low incidence, their occurrence in control fetuses, and/or lack of a dose-response-relationship.
Fetal visceral anomalies observed included levocardia (malrotation of the heart) and hydronephrosis, as well as several other renal developmental variations. Of the levocardia and the hydronephrosis (both classified as malformations), only the former was observed significantly more often in the 2000 mg/kg bw/day group than in the control group. All other findings were classified as variations.

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: increased incidence of levocardia (malrotation of the heart)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: increased incidence of levocardia (malrotation of the heart) at 2000 mg/kg bw/day

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: cardiovascular

Description (incidence and severity):

levocardia (malrotation of the heart); significantly increased at 2000 mg/kg bw/day

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Mean body weights (g)

 

		Dose [mg/kg bw/day]
Day		800	2000	0.0
0	Mean (SD)	239.8 (8.7)	234.9 (10.1)	237.2 (7.3)
3		245.2 (11.7)	234.0 (11.6)**	252.4 (7.6)
6		258.3 (11.4)	250.6 (9.3)**	264.5 (7.0)
10		281.9 (11.7)	270.0 (10.8)**	285.2 (11.6)
13		295.2 (13.9)	284.5 (14.1)*	298.6 (10.4)
16		323.5 (12.5)	311.2 (17.5)	322.2 (10.4)
20		385.5 (17.3)	366.1 (21.7)*	386.0 (13.5)

significantly different from control: * p<0.05, ** p<0.01

 

 

Table 2: Serum chemistry data

 

Serum parameters [unit]	Dose [mg/kg bw/day]
	800		2000		0.0
	Mean (SD)	%	Mean (SD)	%	Mean (SD)
Glucose [mg/dL]	105.2 (13.8)		102.7 (9.3)*	-10	114.0 (12.3)
ALT [IU/L]	46 (8)		51 (7)*	+19	43 (7)
Alk. Phosphatise [IU/L]	118 (46)		140 (55)*°	+50	93 (31)
Creatinine [mg/dL]	0.63 (0.07)*	-7	0.61 (0.04)*	-10	0.68 )0.05)
Cholesterol [mg/dL]	72.6 (15.1)		59.4 (8.2)*°	-25	79.4 (10.5)
Triglycerides [mg/dL]	270.7 (83.9)		204.5 (58.8)*	-39	335.3 (123.3)
Total protein [mg/dL]	5.0 (0.4)*	-6	4.8 (0.2)*°	-9	5.3 (0.3)
Globulin [g/dL]	2.3 (0.3)*	-8	2.2 (0.1)*°	-12	2.5 (0.1)
Iron [µg/dL]	106 (35)		141 (53)*	+52	93 (26)

significantly different from control: * p<0.05; ° outside of historical control range

 

Table 3: Summary of visceral malformations

 

Group		Dose [mg/kg bw/day]
		800	2000	0.0
Number examined	Litters	13	13	15
	Fetuses	94	93	108
Number normal [%]	Litters	7 (53.8%)	3 (23.1%)*	10 (66.7%)
	Fetuses	88 (93.6%)	72 (77.4%)*	102 (94.4%)
Levocardia	Litters	5 (38.5%)	10 (76.9%)*	5 (33.3%)
	Fetuses	5 (5.3%)	21 (22.6%)*	6 (5.6%)
Hydronephrosis, right	Fetuses	1 (7.7%)	0 (0%)	0 (0%)
	Litters	1 (1.1%)	0 (0%)	0 (0%)
Hydronephrosis, left	Fetuses	0 (0%)	2 (15.4%)	0 (0%)
	Litters	0 (0%)	2 (2.2%)	0 (0%)
Hydronephrosis, bilateral	Fetuses	0 (0%)	1 (7.7%)	0 (0%)
	Litters	0 (0%)	1 (1.1%)	0 (0%)

significantly different from control: * p<0.05

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study report, comparable to OECD guideline study with acceptable restrictions (non-GLP, no data on analytical purity)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(adopted 2001)

Deviations:

yes

Remarks:

only one dose level tested instead of three as recommended by guideline; no examinations for skeletal abnormalities performed, no data on test substance purity

Principles of method if other than guideline:

The focus of this study was fetal heart development.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Details on strain: VAF/Plus Crl:CD(SD)BR
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: control group: 231.0 ± 10.1 g; test substance-treated group: 231.3 ± 10.3 g; negative control group: 235.2 ± 11.8 g
- Fasting period before study: no
- Housing: individually
- Diet: Purina Certified Rodent Chow #5002 (Meal); ad libitum
- Water: tap water, via an automatic watering system; ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: clipped, intact dorsal skin
- Type of wrap: open application, no wrap used
- Time intervals for shavings or clipplings: on gestation day 0 and once weekly thereafter

REMOVAL OF TEST SUBSTANCE
- Washing: no washing

TEST MATERIAL
- Constant volume or concentration used: yes
- The test material was measured using a 1.00 mL syringe (calibrated in 0.01 mL) for both groups, and during dispensing was spread evenly on the clipped dorsal skin of the rats using the tip of the syringe. (No needle was used.)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
To minimize ingestion of the test material, the rats were fitted with cardboard Elisabethan-style collars. These collars were lined with latex tubing to minimize the development of irritation or lesions.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- If cohoused: cohoused with male rats from the same source
- M/F ratio per cage: 1:1
- Duration of mating period: 10 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

gestation days 0-19

Frequency of treatment:

once daily

Duration of test:

20 days

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

other: negative control substance (CRU No.: #85018)

Details on study design:

- Dose selection rationale:
In the present study, the test substance was administered to presumed-pregnant rats using the same route (dermal) and exposure level (2000 mg/kg bw/day) as in a previous study in which an increased incidence of levocardia in the exposed fetuses was observed. Another substance was chosen for use as a negative control since it has been used in a previous developmental toxicity study and is known to have no adverse effects on the developing conceptus. The primary objectives of this follow up study were (1) to reproduce the findings observed in the rat fetuses in the previous study, (2) to compare the sensitivity of two visceral examination procedures in detecting anomalous heart development, and (3) to determine what effect, if any, the abnormal heart development would have on pup survival.

The study was designed to be performed in two phases: Prenatal and Postnatal.
In the prenatal phase, fetuses were removed by caesarean section on gestation day 20 and examined for the presence of heart anomalies using two procedures: sectioning by razor blade (Wilson's technique) and in vivo dissection (Staple's technique). The postnatal phase was designed to determine the effects of anomalous heart development on pup survival and was to be initiated only if heart anomalies were observed in the prenatal phase. Since the prenatal results were negative, the postnatal phase was not performed.

- Dermal control: Presumed-pregnant rats were clipped as the animals from the treatment groups. The intact dorsal skin of each rat was stroked with the tip of a 1.00 mL syringe, but no test material was applied.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: mortality, clinical signs of ill-health (pathosis), appearance, abortion, premature delivery, behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 6, 10, 16, and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: The thoracic and abdominal cavities were opened and the reproductive organs were examined grossly for evidence of pathological abnormalities.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Parameters: gender, body weight, length, gross external examination

After gross external evaluation, each fetus was immediately placed in an individual compartment of a partitioned plastic container filled with ice water (2 - 9°C) for at least 5 min.

- Soft tissue examinations: Yes: all per litter
- Half of the fetuses were grossly examined for visceral anomalies using Staples technique. The remaining fetuses were placed in Bouin's solution and were evaluated using Wilson's technique of sectioning by razor blade.

- Skeletal examinations: No
- Head examinations: No

Statistics:

Maternal biophase and caesarean section data, and fetal data were evaluated by analysis of variance followed by group comparisons using Fisher's Exact or Dunnet's Test. Fetal visceral data were evaluated statistically by ANOVA followed by group comparisons using Bartlett's Test.
Differences between control and treated groups were considered statistically significant if the probability of the difference being due to chance was less than 5% (p<0.05).

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Reduced body weight gains

Details on maternal toxic effects:
- Observations during gestation:
Red nasal exudate, chromodacryorrhea and neck lesions were observed equally in control and treated animals. According to the study author, these are common findings in animals that were collared.
Signs of dermal irritation, including erythema, edema, flaking of the skin, and scabbing, were observed in most of the animals from the treatment group and the negative control group. In general, the irritation was mild.

- Maternal body weights:
Test substance-exposed and negative control animals gained significantly less body weight throughout the study than control animals. However, this was not considered to be an adverse effect for the dams.

- Observations of dams at caesarean section:
At the time of necropsy, no findings attributable to exposure to the test substance were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed during the study

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
- Fetal evaluations:
Fetal body weights were not significantly affected by exposure to the test substance or the negative control.
No malformations or variations attributable to exposure to the substance or the negative control were observed at the time of external examination of fetuses. One fetus in the control group and one fetus in the test substance-exposed group had microphthalmia (left eye only). One fetus in the negative control group was edematous and had anophthalmia (left eye) and brachdactyly (bilateral hindpaws). Another fetus in this same group had a shortened and filamentous tail. None of these findings were considered to be treatment related.
During gross visceral examination the following findings were recorded: One fetus in the test substance-exposed group had moderate dilation of the renal pelvis. One fetus in the negative control group had a common truncus arteriosus (common aortic and pulmonary trunk) and another fetus in this group had situs inversus of the heart, vessels, lungs, and stomach.
Anomalies observed at the time of visceral examination by sectioning included dilation of the lateral ventricles of the brain, anophthalmia, microphthalmia, and dilation of the renal pelvis. Only dilation of renal pelvis was noted in the test substance-exposed group; all other findings were in the control group. There were no findings for the negative control group.
None of the above mentioned visceral findings were considered to be test material related since they occurred as isolated incidences or were seen in control fetuses.
No evidence for levocardia was seen in this study.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed during the study

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Mean body weights (g)

 

		Dose [mg/kg bw/day]
Day		2000	Negative control	0.0
0	Mean (SD)	231.3 (10.3)	235.2 (11.8)	231.0 (10.1)
6		246.5 (11.7)**	256.8 (14.4)	264.5 (12.7)
10		261.8 (11.6)**	272.2 (17.9)	281.4 (14.6)
16		299.3 (15.5)**	312.1 (19.8)*	324.0 (16.3)
20		350.2 (21.3)**	364.8 (27.7)*	382.4 (20.6)

significantly different from control: * p<0.05, ** p<0.01

Table 2: Summary of visceral findings (Modified Staples’ Technique)

 

Group		Dose [mg/kg bw/day]
		2000	Negative control	0.0
Number examined	Litters	24	25	25
	Fetuses	163	165	178
Vessels: Common truncus arteriosis	Litters	0	1 (4.0%)	0
	Fetuses	0	1 (0.6%)	0
Heart, vessels, lungs, stomach: situs inversus	Litters	0	1 (4.0%)	0
	Fetuses	0	1 (0.6%)	0
Kidney(s): moderate dilation of perlvis	Fetuses	1 (4.2%)	0	0
	Litters	1 (0.6%)	0	0

significantly different from control: * p<0.05

 

Table 3: Summary of visceral findings (’s Sectioning)

 

Group		Dose [mg/kg bw/day]
		2000	Negative control	0.0
Number examined	Litters	24	25	25
	Fetuses	157	152	168
Brain: slight dilation of lateral ventricles	Litters	0	0	2 (8.0%)
	Fetuses	0	0	2 (1.2%)
Eyes: depressed bulge	Litters	0	0	2 (8.0%)
	Fetuses	0	0	2 (1.2%)
Eyes: anophthalmia	Litters	0	0	1 (4.0%)
	Fetuses	0	0	1 (0.6%)
Eyes: microphthalmia	Litters	0	0	1 (4.0%)
	Fetuses	0	0	1 (0.6%)
Kidneys: slight to moderate dilation of pelvis	Litters	2 (8.3%)	0	1 (4.0%)
	Fetuses	2 (1.3%)	0	1 (0.6%)

significantly different from control: * p<0.05

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Historical control data:

yes

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: The decrease in body weight seen at 2000 mg/kg bw/d was not considered to be an adverse effect.

Remarks on result:

other: source: CAS 16958-92-2

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: see 'Remarks'

Remarks on result:

other: source: CAS 16958-92-2

Key result

Abnormalities:

no effects observed

Localisation:

visceral/soft tissue: cardiovascular

Description (incidence and severity):

The increased incidence of levocardia (malrotation of the heart) in the original developmental toxicity study (Exxon, 1988) observed at 2000 mg/kg bw/day could not be confirmed in a second study, focused on heart development (Exxon, 1990). In addition, this study also revealed that there were no other heart defects associated with exposure to the test material. Thus, it could be demonstrated that the test substance is not teratogenic and that the previously observed "levocardia" was not a sign of fetotoxicity of the substance. Therefore, the NOAEL of developmental toxicity is 2000 mg/kg bw/day.

Key result

Developmental effects observed:

no

Conclusions:

In a dermal developmental toxicity study (Exxon, 1988) with the source substance Ditridecyl adipate (CAS 16958-92-2) the NOAEL for maternal toxicity in Sprague Dawley rats was set at ≥ 2000 mg/kg bw/day. For developmental toxicity, a NOAEL of 800 mg/kg bw/day was derived based on increased incidence of levocardia (malrotation of the heart) at 2000 mg/kg bw/day. The increased incidence of levocardia could, however, not be confirmed in a second study (Exxon, 1990). In addition, this study also revealed that there were no other heart defects associated with exposure to the test material. Thus, it could be demonstrated that the test substance is not teratogenic and that the previously observed "levocardia" was not a sign of fetotoxicity of the substance. Therefore, the NOAEL for developmental toxicity is > 2000 mg/kg bw/day. As explained in the analogue justification, these results are considered to be valid also for the target substance.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study from an analogue source substance. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to analogue justification for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from an analogue source substance. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to analogue justification for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

Additional information

Justification for read-across

There are no experimental data on developmental toxicity available for Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3). To fulfil the standard data requirements defined in Regulation (EC) No. 1907/2006, Annex VIII, 8.7 read-across from appropriate substances is conducted in accordance with Regulation (EC) No. 1907/2006, Annex XI.

According to 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) “to avoid the need to test every substance for every endpoint”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read- across approach is provided in the technical dossier (see IUCLID Section 13).

As no experimental data are available on developmental toxicity, read-across of reliable data on the analogue substances Dibutyl adipate (CAS 105-99-7) and Ditridecyl adipate (CAS 16958-92-2) was conducted.

Oral

CAS 105-99-7

An oral reproduction/developmental toxicity screening test with Dibutyl adipate (CAS 105-99-7) was performed in Sprague-Dawley rats according to OECD 421 and in compliance with GLP, described in detail under reproduction toxicity (MHLW, 1996, WoE). Based on the results of this study, the NOAEL for systemic toxicity in parental animals was set at 300 mg/kg bw/day, similar to the NOAEL of the F1 generation, which is based on slight, although statistically significant decrease (96.1%) in the pup viability on Day 4 of lactation at the 1000 mg/kg bw/day dose group compared with controls (100%).

 

Dermal

CAS 16958-92-2

The prenatal developmental toxicity of Bis(tridecyl) adipate (CAS 16958-92-2) via the dermal route was investigated in a study similar to OECD 414 (Exxon, 1988, WoE). Groups of 15 pregnant Sprague Dawley rats were exposed dermally to the test substance at dose levels of 800 and 2000 mg/kg bw/day. A similar constituted group of females was sham-exposed and served as control. The undiluted test substance was applied once daily to the clipped dorsal skin of the animals during Days 0 to 19 of gestation under open conditions. On Day 20 of gestation, dams were sacrificed and maternal as well as fetal examinations were performed. Only slight maternal toxicity was observed at 2000 mg/kg bw/day as indicated by a non-adverse, but significantly reduced gain in body weight compared to controls during the first and last interval of gestation, respectively. Although body weights were significantly lower at this dose level, no corresponding change in overall food consumption was noted at the end of the study compared to controls. Statistically significant differences between the serum clinical chemistry parameters from control and treated rats were observed for glucose, alanine aminotransferase (ALT), alkaline phosphatase, creatinine, cholesterol, triglycerides, total protein, iron, and globulin. However, these changes were not considered biologically significant, since they were not accompanied by any particular findings in the dams. Mild dermal irritation, including erythema and flaking of the skin, were observed in most of the animals at 800 and 2000 mg/kg bw/day. Scabs were observed at the side of test substance application in two animals exposed to 800 mg/kg bw/day. No adverse effects were observed for female reproductive parameters (number of implants, resorptions or viable fetuses). Mean fetal body weights and crown-rump lengths were not affected by exposure to the test substance. Fetal examination did not reveal any treatment-related effects on skeletal development. Although one fetus exposed in utero to 2000 mg/kg bw/day showed multiple external malformations, the effects were not considered to be a result of exposure to the test substance. Fetal visceral anomalies observed included levocardia (malrotation of the heart) and hydronephrosis as malformations after exposure to the test substance. However, only levocardia was significantly increased in the 2000 mg/kg bw/day group compared to controls. Based on the results of the study, the NOAEL for maternal toxicity in Sprague Dawley rats was set at ≥ 2000 mg/kg bw/day. For developmental toxicity, a NOAEL of 800 mg/kg bw/day was derived.

In a subsequent heart developmental toxicity study similar to OECD 414, the test substance was dermally applied to the clipped skin of 25 female Sprague Dawley rats at a limit dose of 2000 mg/kg bw/day during Days 0 to 19 of gestation (Exxon, 1990, WoE). Two additional groups of each 25 pregnant females were sham-exposed or treated with a negative control substance and served as control or negative control group, respectively. The dams were sacrificed on Day 20 of gestation and maternal and fetal examinations were performed. Mild dermal irritation, including erythema, edema, flaking of the skin, and scabbing, was observed in most of the animals from the treatment group and the negative control group. In the test substance-exposed and negative control animals, a statistically significant reduction in body weight was noted throughout the study compared to controls. However, this effect was not considered to be adverse. In addition, female reproductive parameters were not adversely affected by treatment with the test substance and the negative control, respectively. Determination of fetal body weights as well as external examination, and visceral examinations of foetuses did not reveal any substance-related effects. Visceral findings either occurred sporadically or were also observed in the sham-exposed control group. Heart development was not adversely affected by treatment with the test substance, and did thus not confirm the occurrence of levocardia as observed in the previous prenatal toxicity study. Based on the results of this study, the NOAEL for maternal and developmental toxicity in Sprague Dawley rats was greater than 2000 mg/kg bw/day after dermal application of Bis(tridecyl) adipate.

 

Conclusion for developmental toxicity

In summary, an oral reproduction/developmental toxicity screening test is available with the source substance Dibutyl adipate (CAS 105-99-7), which did not show signs of developmental toxicity in the absence of maternal toxicity. In addition, in a dermal developmental toxicity studies with the source substance Bis(tridecyl) adipate (CAS 16958-92-2) the NOAEL for dermal developmental toxicity was established at 800 mg/kg bw/day based on the incidence of levocardia at 2000 mg/kg bw/day. However, the adverse effects of Bis(tridecyl) adipate were not confirmed in a subsequent heart developmental toxicity study via the dermal route, resulting in a NOAEL for developmental toxicity of ≥ 2000 mg/kg bw/day. Thus, as the available data did not identify any hazard for toxicity to development, Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) is not expected to be hazardous following oral or dermal exposure.

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on reproduction and developmental toxicity do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.