Fatty acids, C18-unsatd, dimers, hydrogenated, diisopropyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental 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

Study period:

12 May - 23 June 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance fatty acids, C18-unsatd., dimers (CAS No. 61788-89-4). In accordance to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Rat/IGS (Crl: CD®(SD) IGS BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: ca. 8 weeks
- Weight on arrival: (P) Males: 145 - 165 g; (P) Females: 102 - 138 g
- Housing: Initially animals were housed 2 per polypropylene cage (42 x 72 x 20 cm) with stainless steel grid bottoms and mesh tops. A few days prior to pairing, males were transferred to individual grid-bottomed cages (58 x 38.5 x 20 cm). Mated females were housed individually in solid-bottomed cages (42 x 72 x 20 cm).
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 Expanded Ground SQC (Special Diets Services Ltd., Stepfield, Witham, Essex, UK), ad libitum
- Water (e.g. ad libitum): domestic quality mains water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 12 May 2003 To: 23 June 2003

Route of administration:

oral: feed

Vehicle:

other: not applicable

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of diet were prepared at convenient intervals (weekly or fortnightly) during the study, and used within 12 days of preparation.
- Mixing appropriate amounts with (Type of food): An appropriate quantity of the test item was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet and mixed for ca. 1 h with fan assisted venting to aid removal of the acetone. This dose premix was diluted with untreated diet to give the diet for the intermediate and high dose groups. The low dose diet was prepared by dilution of the intermediate dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for ca. 20 min.
A control premix was prepared in the same way as the dose premix by using acetone and untreated diet. By dilution of this control premix with untreated diet the control diet was made, containing the same proportion of premix as the high dose diet.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight or up to 16 days
- Proof of pregnancy: copulatory plug and/or sperm in the lavage referred to as day 0 of pregnancy
- After 9 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulated diets were undertaken on 2 occasions during study treatment with regard to concentration and homogeneity. On each occasion triplicate samples of 50 g were withdrawn from each formulated diet (low, mid and high dose diet and control diet). The samples were analysed by the Toxicology Support Laboratory using a method supplied by the sponsor (no further data on method). ± 15% of the nominal concentration indicated an acceptable accuracy of formulation.

Results (mean found) of first analysis (in parenthesis: % difference from nominal):
200 ppm: 176 ppm (- 12%)
2000 ppm: 1770 ppm (- 11.5%)
20000 ppm: 16849 ppm (- 15.8%); reanalysis: 20862 ppm (+ 4.3%)

Results (mean found) of second analysis (in parenthesis: % difference from nominal):
200 ppm: 155 ppm (- 22.5%); reanalysis: 173 ppm (- 13.5%)
2000 ppm: 2109 ppm (+ 5.5%)
20000 ppm: 20609 ppm (+ 3.0%)

The low coefficient of variation (5.6% or less) was indicative of satisfactory homogeneity in all dose groups.

Duration of treatment / exposure:

(P) Males: at least 4 weeks overall, starting from 2 weeks prior to mating until termination
(P) Females: 2 weeks prior to mating, then through mating until at least day 4 of lactation

Frequency of treatment:

continuously

Details on study schedule:

no mating of F1 generation (not foreseen according to OECD 421)

Remarks:

Doses / Concentrations:
0, 200, 2000, 20000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
14.5, 147, 1450 and 16.5, 166, 1692 mg/kg bw/day for males and females, respectively
Basis:
other: group mean achieved dosages of test item calculated on food consumption, nominal dietary concentration and body weight (worst-case assumption of doses)

No. of animals per sex per dose:

10 P males and 10 P females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Existing relevant toxicological data was evaluated for dose selection. Dose levels take into account the maximum tolerable dose in the test animals and other factors such as anticipated exposure.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: damaged teeth, piloerection, staining on dorsal neck/head, abnormal vocalisation, unkempt coat, body held low, hairloss, scabbing on head/mouth, agitation, hunched posture, rolling gait, pale discoloured skin on the whole body etc.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals: one week prior to the start of treatment; Males: weekly throughout the treatment period until termination; Females: weekly until the start of mating period, then on day 0 of gestation, followed by day 7, 14 and 20 of gestation and day 1 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was determined as follows:
All animals: weekly until mating, starting one week prior to start of treatment; Males: weekly measurements continued over complete period, except during periods of co-habitation with females; Mated females: over the periods days 0-7, 7-14 and 14-20 of gestation and days 0-4 of lactation.
The achieved intake of the test item (mg/kg bw/day) was calculated from the following formula: achieved intake = (food consumption x dietary concentration, nominal) / body weight.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Once weekly throughout the study, the water consumption was monitored by visual inspection of the water bottles.

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated in P females by vaginal smears commencing on the day of pairing until mating had occured.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of externally visible anomalies, body weight (day 1 and day 4 per sex and litter), physical or behavioural abnormalities, presence of milk in the stomach

GROSS EXAMINATION OF DEAD PUPS:
No for internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals (P): All surviving animals, when mating has been completed and the animals have been dosed for at least 4 weeks.
- Maternal animals (P): All surviving animals, when all observations have been completed, normally between day 4 and day 7 of lactation.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology was conducted on the epididymides, testes and ovaries of all control and high dose animals. Male reproductive organs (epididymides and testes) were weighed individually.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed between day 4 and day 7 of lactation.
- These animals were not subjected to postmortem examinations (macroscopic and/or microscopic examination).

Statistics:

Kruskal-Wallis non-parametric analysis (body weight, food consumption); analysis of variance and one-way analysis of covariance (organ weights);
Incidence data: Kruskal-Wallis analysis, Chi-squared test or Fisher's Exact Probability test;
Pairwise comparison (against control group): Fisher's F-protected LSD method, Student's t-test or Chi-squared protected z-test;
All statistical tests were two-sided and performed at the 5% significance level.

Reproductive indices:

fertility index (male) = number siring a litter / number paired;
fertility index (female) = number pregnant / number paired;
gestation index = number bearing live pups / number pregnant;

Offspring viability indices:

birth index = total number of pups born (live and dead) / number of implantation scars;
live birth index = number of pups live on day 0 of lactation / total number born (live and dead);
viability index = number of pups live on day 4 of lactation / number live on day 0;

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 2000 ppm dose group two female rats died prematurely. One animal was found dead after displaying several clinical signs, which included staining, piloerection, rolling gait, subdued behaviour, pale discoloured skin and a swollen/damaged tail. The other animal was killed prematurely due to a prolapse of the vagina. This female rat also showed hunched posture, pale eyes, subdued bahaviour and pale discoloured skin on the whole body. These two deaths were considered to be incidental.
Piloerection was noted in 1/10, 5/10, 3/10 and 8/10 males and in 3/10, 2/10, 1/10 and 2/10 females at dose levels of 0, 200, 2000 and 20000 ppm. Given the distribution of these findings and the lack of any dose relationship, it is not possible to associate them with the treatment.
The clinical observations for the other animals were considered to be consistent with those normally seen in rats of this age and strain.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A statistically non-significant decrease in body weight was noted in males of the high dose group (313 ± 18 g) during the first week of treatment compared to the control males (331 ± 22 g). Weight gain thereafter was essentially similar to the control group (table 1). Among females of the high dose group, mean body weight gain was comparable to the controls throughout the study.
At 2000 ppm, there was a slight reduction in body weight gain in males. This minor difference in body weight performance was considered to reflect the low body weight noted at the start of treatment. No obvious effects of treatment in males at 200 ppm or in females at 2000 ppm and 20000 ppm were observed.


FOOD CONSUMPTION (PARENTAL ANIMALS)
In females treated with 20000 ppm mean food consumption was slightly increased during days 0-4 of lactation (29.2 g) compared to control (26.5 g). However, this increase was not significant. In males treated at this level, mean food consumption was essentially similar to control animals (table 1). At 2000 ppm there was a statistically significant increase in food consumption in female rats during the first week of treatment (controls: 18.2 ± 2.0 g; 2000 ppm: 20.2 ± 0.6 g). Thereafter food consumption was comparable to controls throughout the study (table 1). Since the increase in food consumption in female rats at 2000 ppm was not dose-dependent, this finding was not considered to be attributable to the administration of the test substance.
Mean food consumption in the low dose group was similar to that of control animals.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The male and female fertility index was 100% among all dose group. The median number of nights to a positive mating sign was, 2, 2, 4, and 3 nights for 0, 200 ppm, 2000 ppm, 20000 ppm, respectively. There were no animals passing one estrus to positive mating. The mean duration of gestation did not differ between control and treatment groups. Thus, there were no effects of treatment on mating performance, fertility or the duration of gestation.


ORGAN WEIGHTS (PARENTAL ANIMALS)
The testes and epididymides weights were essentially similar in all groups (table 1).


GROSS PATHOLOGY (PARENTAL ANIMALS)
In the female rat of the 2000 ppm dose group found dead all tissues were autolysed and the left horn of the uterus was enlarged. In another female of the same dose group killed prematurely due to a prolapse of the vagina a small thymus was examined.
Pelvic dilation in the right kidney in one female rat of the low dose group and distended urinary bladder in one male rat of the low dose group were considered not to be treatment-related. No further necropsy findings were noted in any of the dose groups.


HISTOPATHOLOGY (PARENTAL ANIMALS)
No abnormalities were noted by the examination of ovaries in the control and high dose females.
At the examination of testes, minimal seminiferous epithelial degeneration (bilateral) was observed in one male of the high dose group. No abnormalities in testes were seen in control rats. At the examination of the epididymis in the high dose group and control group chronic inflammatory cell infiltration was noted in three treated animals and in one control animal, respectively. In another control animal chronic inflammation (focal, adnexal) of the epididymis were seen. Minimal cellular debris (luminal, bilateral) was identified in one male at 20000 ppm. However, these findings in testes and epididymis were considered not to be treatment-related.


OTHER FINDINGS (PARENTAL ANIMALS): ACHIEVED DIETARY INTAKE
The achieved dietary intake for females in the high dose group was lower in the first week (1886 mg/kg bw/day) than in the second week (2190 mg/kg bw/day). Among males treated with 20000 ppm a higher mean dietary intake was achieved during the first week (1889 mg/kg bw/day) than in the following weeks (week 2: 1875 mg/kg bw/day; week 4: 1450 mg/kg bw/day).
In addition, there was a decreased concentration in females throughout the gestation period in all treatment groups (table 1). During this period, the achieved concentrations at all levels were slightly less than proportional to the diet concentrations.
At other times, the achieved concentration was essentially proportional to the diet concentrations.

Dose descriptor:

NOAEL

Effect level:

>= 1 450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: achieved intake equivalent to 20000 ppm (worst-case assumption); Basis: clinical signs; mortality; body weight; food consumption and compound intake; gross pathology; organ weights; histopathology; fertility index;

Dose descriptor:

NOAEL

Effect level:

>= 1 692 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

>= 1 692 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: achieved intake equivalent to 20000 ppm (worst-case assumption); birth index; live birth index; litter size; litter weight; pup weight; viability index; grossly visible abnormalities

Remarks on result:

other: Generation: developmental toxicity (migrated information)

VIABILITY (OFFSPRING)
The mean number of implants per pregnancy was higher in all the treated groups compared to the control (table 2). However, historical data shows that the findings in the treated groups were within background ranges for animals of this age and strain in similar studies performed at the testing laboratory. It was considered most likely, that the control value is at the lower end of the background range and thus, these findings were not treatment-related.
No obvious effects of treatment on litter size or litter survival in any of the dose groups were noted (table 2).

BODY WEIGHT (OFFSPRING)
There were no obvious effects of treatment on litter or pup weights in any of the dose groups (table 2).

OTHER FINDINGS (OFFSPRING)
There were no externally visible anomalies detected among all pups.

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Table 1: Reproductive/developmental toxicity screening test: parental examinations

	Control group	200 ppm	2000 ppm	20000 ppm
Body weight gain (g)
Males Week 0	288 ± 18	284 ± 14	277 ± 11	282 ± 15
Males Week 1	331 ± 22	323 ± 12	320 ± 17	313 ± 18
Males Week 2	370 ± 29	374 ± 18	358 ± 22	361 ± 24
Males Week 3	404 ± 36	409 ± 19	387 ± 26	394 ± 29
Males Week 4	432 ± 37	437 ± 23	411 ± 29	417 ± 39
Males Week 0-4	144 ± 22	153 ± 13	134 ± 21	135 ± 25
Females Week 0-2	41 ± 12	43 ± 8	38 ± 7	37 ± 7
Females Day 0-20 of gestation	139	143	140	146
Females Day 4 of lactation	287 ± 18	290 ± 12	294 ± 20	293 ± 22
Food consumption (g)
Females Week 0	21.0 ± 1.2	18.0 ± 3.9	18.2 ± 2.0	18.2 ± 2.1
Females Week 1	18.1 ± 2.8	17.9 ± 0.5	20.2 ± 0.6 **	19.1 ± 0.8
Females Week 2	24.3 ± 3.2	21.5 ± 2.2	22.3 ± 1.9	24.2 ± 0.9
Females Day 0-4 of lactation	26.5	22.7	27.9	29.2
Group mean achieved dosages of test item (mg/kg bw/day)
Males Week 1	-	18.6	201	1889
Males Week 2	-	18.4	177	1875
Males Week 4	-	14.5^1	147^1	1450^1
Females Week 1	-	18.4	202	1886
Females Week 2	-	19.9	204	2190
Females Day 0-7 of gestation	-	19.4	189	1980
Females Day 7-14 of gestation	-	18.6	184	1836
Females Day 14-20 of gestation	-	16.5^1	166^1	1692^1
Females Day 1-4 of lactation	-	16.6	199	2097
Absolute epididymides weights (g)
Males	1.1258 ± 0.0809	1.1205 ± 0.0548	1.0607 ± 0.0611	1.0555 ± 0.1157
Absolute testes weights (g)
Males	3.39 ± 0.16	3.48 ± 0.32	3.36 ± 0.24	3.38 ± 0.35

** significantly different from control P<0.01

¹ : These values represent the “worst-case” achieved intakes, which were taken into account for hazard assessment.

 Table 2: Reproductive/developmental toxicity screening test: Reproductive perormance, litter size and survival, litter and pup weights

	Control group	200 ppm	2000 ppm	20000 ppm
Mean duration of gestation and overall litter performance values
Mean number pregnant	10	10	10	10
Mean duration of gestation (days)	21.7	21.8	21.6	21.7
Number of females producing a liver litter	10	10	9	10
Gestation index (%)	100	100	90	100
Mean number of implant sites^aper pregnancy	12.9± 2.3	15.6 ± 0.8	14.7 ± 2.6	15.2 ± 1.9
Mean total number of pups^aborn per litter	11.8 ± 2.0	14.1 ± 1.6	13.0 ± 1.8	14.1 ± 1.6
Mean number of live pups per litter Day 0 of lactation	11.8 ± 2.0	14.1 ± 1.6	12.9 ± 2.0	13.7 ± 1.8
Mean number of live pups per litter Day 4 of lactation	11.6 ± 2.0	12.1 ± 3.4	12.6 ± 2.1	12.0 ± 3.9
Group mean F1 survival indices
Birth index (%)	91	91	91^b	92
Live birth index (%)	99	100	99^b	93
Viability Index Days 0-4(%)	79	87	86^b	82
Group mean litter and pup weight^a
Litter Day 1 of lactation (g)	78 ± 10	73 ± 22	79 ± 12	82 ± 11
Litter Day 4 of lactation (g)	112 ± 13	105 ± 30	116 ± 18	111 ± 35
Males Day 1 of lactation (g)	7.0± 0.8	6.0 ± 0.9	6.5 ± 0.6	6.4 ± 0.9
Males Day 4 of lactation (g)	10.0 ± 1.3	8.9 ± 1.1	9.5 ± 0.9	9.0 ± 1.1
Females Day 1 of lactation (g)	6.5 ± 0.8	5.6± 0.9	6.2 ± 0.5	6.1 ± 1.0
Females Day 4 of lactation (g)	9.5 ± 1.3	8.5 ± 1.2	9.1 ± 0.7	8.8 ± 1.3

^a: excludes litters where all pups died

^b: based on 8 litters (litters of animal found dead and of animal killed prematurely were not considered)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 2) study from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity between the source and target substances, the source substance being a product of the hydrolysis of the 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 toxicity to reproduction of Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). In order 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, 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 toxicity to reproduction:

CAS	Chemical name	Molecular weight	Toxicity to reproduction	Developmental toxicity / teratogenicity
103213-20-3 (a)	Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters	645	RA: CAS 61788-89-4	RA: CAS 104-76-7
61788-89-4 (b)	Fatty acids, C18-unsaturated, dimers	564	Experimental result: NOAEL (P) ≥ 1450/1692 (females/males) mg/kg bw/day; NOAEL (developmental toxicity) ≥ 1692 (females/males) mg/kg bw/day (rat)	--
104-76-7 (b)	2-ethylhexanol	130	--	Experimental result: NOAEL (maternal/developmental) = 191 (females/males) mg/kg bw/day (mouse)

(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 substances are considered to be either the theoretically possible hydrolysis product (CAS 61788-89-4) or the surrogate substance (CAS 104-76-7) for the other theoretically possible hydrolysis product 2-propanol. The available endpoint information is used to predict the same endpoints for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion

Effects on fertility

No data on toxicity to reproduction is available with Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). Therefore, read across from the possible surrogate hydrolysis product Fatty acids, C18-unsaturated, dimers (CAS61788-89-4) and 2-ethylhexanol (CAS 104-76-7) was applied.

CAS 61788-89-4

A key reproduction/developmental toxicity screening test performed according to OECD TG 421 and in compliance with GLP with Fatty acids, C18-unsaturated, dimers (CAS 61788-89-4) is available (Clubb and Sutherland, 2004). Groups of 10 Sprague-Dawley rats of each sex per dose were administered doses of 200, 2000 and 20000 ppm corresponding to 14.5, 147 and 1450 mg/kg bw/day and 16.5, 166 and 1692 mg/kg bw/day for males and females, respectively of the test substance via the diet. Males were treated for at least 4 weeks, starting from 2 weeks prior to mating; females were treated for 2 weeks prior to mating, during mating and gestation until at least day 4 of lactation. The animals were monitored for clinical signs, body weight, food consumption, mating and litter performance. All animals were submitted to necropsy, which included weighing of testes and epididymides. Histopathology was conducted on male and female reproductive organs of the control and high dose group. The observed effects were limited to the 20000 ppm males and included slightly decreased body weight gain during the first week of treatment and an increased incidence of piloerection. However, weight gain for males at 20000 ppm was essentially similar to control animals for the rest of the study on weeks 2-4 and the incidence of piloerection lacked any dose-relationship. There were no effects on testes, epididymides and ovaries and on reproductive performance between control and test animals. The fertility index was 100% for males and females in all dose groups. A NOAEL (reproductive toxicity/fertility) of ≥ 1450 mg/kg bw/day and ≥ 1692 mg/kg bw/day for males and females, respectively, was determined based on no toxicologically significant effects at the highest dose tested. Moreover, a NOAEL (developmental toxicity) of ≥ 1692 mg/kg bw/day for both sexes was determined. The molecular weight ratio of Fatty acids, C18-unsaturated, dimers (MW 564.93 g/mol) and Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (MW 645.06 g/mol) is 1.14 (645.06/564.93). Thus, based on this molecular ratio factor the NAEL for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3) was calculated to be greater than 1653 and 1928.88 mg/kg bw/day for males and females, respectively (1450 mg/kg bw/day x 1.14; 1692 mg/kg bw/day x 1.14).

Short description of key information:
Reproduction/Developmental Toxicity Screening (OECD 421), rat NOAEL (parental) ≥ 1450/1692 mg/kg bw/day (males/females); NOAEL (developmental) ≥ 1692 mg/kg bw/day (males/females) (RA CAS 61788-89-4), corresponding NAEL (parental) = 1653/1928.88 mg/kg bw/day (males/females); NAEL (developmental) ≥ 1928.88 mg/kg bw/day (males/females) for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from structural analogues/surrogates. 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 levell.

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECD 414), mouse NOAEL (maternal/developmental) = 191 mg/kg bw/day (RA CAS 104-76-7), corresponding NAEL = 945.45 mg/kg bw/day for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters

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

Study period:

1990-05-18 to 1990-08-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to Guideline study, tested with the metabolite 2-ethylhexanol (CAS 104-76-7). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Weight at study initiation: 23.52-31.59 g
- Housing: Plug-positive females were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and Ab-Sorb-Dri® cage litter (Laboratory Products, Garfield, NJ
- Diet (e.g. ad libitum): Ground Purina Certified Rodent ChoW® (#5002) available ad libitum throughout gestation
- Water (e.g. ad libitum): deionized/filtered water were available ad libitum throughout gestation
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2
- Humidity (%): 48
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: food grade modified corn starch microcapsules

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh supplies of dosed feed were obtained from refrigerated stock on the mornings of gd 0, 3, 6, 9, 12 and 15

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis by gas chromatography (GC) prior to use verified the formulations to be within 99-108% of the theoretical concentrations. 2EH/feed mixes were determined to be stable throughout the period of use for each study replicate.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage:1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Verification of same strain and source of both sexes: yes

Duration of treatment / exposure:

continuously exposure to 2-EH (0, 0.009, 0.03, or 0.09%) microencapsulated in the feed from gestational day 0-17

Frequency of treatment:

continuously ad libitum feed

Duration of test:

sacrifice at gestational day 17

Remarks:

Doses / Concentrations:
0.009, 0.03 or 0.09%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 17, 59 and 191 mg/kg bw/d
Basis:
other: calculated consumption, based on gestational food consumption

No. of animals per sex per dose:

28

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: DEHP was previously evaluated for potential developmental toxicity in timed-pregnant Swiss (CD-I) mice exposed via the diet throughout gestation (gd 0 to 17) (Tyl et oZ., 1988). An average dose of 44 mg/kg/day (0.025% DEHP in feed) was the maternal and embryo/fetal NOAEL. An increased incidence of malformations was observed at 91 mg/kg/day (0.05% DEHP in feed) in the absence of other indications of maternal and embryo/fetal toxicity. At 191 and 292 mg/kg/day (0.10% and 0.15% DEHP in feed), maternal toxicity (reduced weight gain during treatment and increased relative liver weight) was observed, as well as decreased fetal weight and an increased incidence of prenatal mortality and fetal malformations. Based upon these findings, additional studies were designed to characterize the developmental toxicity of DEHP's principal metabolites (MEHP and 2-18 ethylhexanol) at approximately equimolar doses and under comparable experimental conditions as those from the study of DEHP in mice (Tyl et aZ., 1988). Accordingly, the concentrations of MEHP in feed included 0% (control), 0.017%, 0.035%, 0.070%, and 0.140%, with the average daily intake of 0, 35, 73, 134, and 269 MEHP mg/kg/day, respectively (NTP, 1990), approximately equivalent on a molar basis to the dose levels of DEHP used by Tyl et aZ. (1988). The target concentrations of 2-EH in feed employed for this study included 0.00% (control), 0.009%, 0.030%, and 0.090%. The expected average daily intake of 2-EH at the proposed dietary concentrations were 0, 15, 52.5, and 157.5 mg/kg/day, respectively. (The actual intake was 0, 17, 59 and 191 mg/kg/day; see results.) Therefore, the target dietary dose levels of 2-EH employed for this study were intended to encompass the range of intakes obtained with DEHP.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from gestation day 0-17

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 3, 6, 9, 12, 15, and 17

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: on gestation days 0, 3, 6, 9, 12, 15, and 17

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: The maternal body, liver, and intact uterus were weighed

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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

analyses of variance (ANOVA), when ANOVA revealed a significant (p<0.05) dose effect, Dunnett's Multiple Comparison Test (Dunnett, 1955; 1964) compared each 2-EH-exposed group to the control group. One-tailed tests were used for all pairwise comparisons except maternal body and organ weights and fetal body weight, General Linear Models (GLM), chi square test, when a chi square test showed significant group differences, a one-tailed Fisher's exact probability test was used for pairwise comparisons of each 2-EH group with the control group.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No females died, delivered early or were removed from the study. Pregnancy rates were high and equivalent across all groups (93-96%); maternal
weight change for the gestational (and treatment) period, gd 0-17, was unaffected, as was weight change corrected for gravid uterine weight; maternal organ weights and food consumption were also unaffected; Treatment related clinical signs of toxicity were limited to hyperactivity observed in
one dam on gd 6, 9 and 12 at 0.090% and in one dam on gd 6 at 0.030%

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of exposure on the number of ovarian corpora lutea, or of uterine implantation sites (resorptions, dead fetuses or live fetuses) per litter. Live litter size and fetal body weight per litter (all fetuses, males or females) were equivalent across all groups.
There were also no effects of treatment on the incidence of malformations (external, visceral, skeletal or total) or variations, whether expressed as number or percentage of fetuses per litter or of litters with one or more affected fetuses. Examination of individual fetal findings also indicated no specific malformations or variations with a dose-related incidence.

Dose descriptor:

NOAEL

Effect level:

191 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects on: litter size and weights; number viable (number alive and number dead); sex ratio; grossly visible abnormalities; external, soft tissue and skeletal abnormalities;

Abnormalities:

not specified

Developmental effects observed:

not specified

In conclusion, 2-EH administered in the diet during gestation (gd 0-17) in CD1 mice at concentrations comparable to or exceeding those employed for DEHP and MEHP in the same study design, resulted in no maternal or developmental toxicity. It is therefore clear that 2-EH plays essentially no role in the expression of DEHP-induced maternal and developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

191 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

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

Justification for grouping of substances and read-across

There are only limited data available on toxicity to reproduction of Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). In order 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, 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 toxicity to reproduction:

CAS	Chemical name	Molecular weight	Toxicity to reproduction	Developmental toxicity / teratogenicity
103213-20-3 (a)	Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters	645	RA: CAS 61788-89-4	RA: CAS 104-76-7
61788-89-4 (b)	Fatty acids, C18-unsaturated, dimers	564	Experimental result: NOAEL (P) ≥ 1450/1692 (females/males) mg/kg bw/day; NOAEL (developmental toxicity) ≥ 1692 (females/males) mg/kg bw/day (rat)	--
104-76-7 (b)	2-ethylhexanol	130	--	Experimental result: NOAEL (maternal/developmental) = 191 (females/males) mg/kg bw/day (mouse)

(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 substances are 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 Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion

Effects on developmental toxicity

No data on toxicity to reproduction is available with Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3). Therefore, read across from the possible surrogate hydrolysis product Fatty acids, C18-unsaturated, dimers (CAS61788-89-4) and 2-ethylhexanol (CAS 104-76-7) was applied.

CAS 104-76-7

A reliable key developmental toxicity study performed equivalent or similar to OECD TG 414 with 2-ethylhexanol (CAS 104-76-7) is available (Tyl, 1991). Groups of 28 CD-1 mice were treated with modified corn starch microcapsules in the diet continuously from 0 to day 17 of gestation with 0.009, 0.03 or 0.09% in feed corresponding to 17, 59 and 191 mg/kg/day of the test substance. No females died, delivered early or were removed from the study. Pregnancy rates were high and equivalent across all groups (93-96%), maternal weight change for the gestational (and treatment) period, gestation day 0-17, was unaffected, as was weight change corrected for gravid uterine weight and maternal organ weights and food consumption were also unaffected. There were no effects of exposure on the number of ovarian corpora lutea, or of uterine implantation sites (resorptions, dead fetuses or live fetuses) per litter. Live litter size and fetal body weight per litter (all fetuses, males or females) were equivalent across all groups. There were also no effects of treatment on the incidence of malformations (external, visceral, skeletal or total) or variations, whether expressed as number or percentage of fetuses per litter or of litters with one or more affected fetuses. Examination of individual fetal findings also indicated no specific malformations or variations with a dose-related incidence. In conclusion, the test material administered in the diet during gestation days 0-17 in CD1 mice at concentrations of 17, 59 and 191 mg/kg bw/day resulted in no maternal or developmental toxicity. Therefore, the NOAEL for developmental toxicity was considered to be 191 mg/kg bw/day. The molecular weight ratio of 2-ethylhexanol (MW 130.23 g/mol) and Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (MW 645.06 g/mol) is 4.95 (645.06/130.23). Thus, based on this molecular ratio factor the NAEL for Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters (CAS 103213-20-3) was calculated to be 945.45 mg/kg bw/day (191 mg/kg bw/day x 4.95).

Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from structural analogues/surrogates. 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.

Justification for classification or non-classification

The available data on reproductive/developmental toxicity of Fatty acids, C18-unsatd., dimers, hydrogenated, diisopropyl esters do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.