Diisodecyl sebacate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study on a read-across compound, reported in the secondary literature (limited reporting). Only one (relatively low) dose level tested (about 10 mg/kg bw/day); screening reproduction/developmental screening limit tests use at least 1000 mg/kg bw/day.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four-generation study, in which rats were treated via the diet with DOS at 200 mg/kg. Only one (relatively low) dose level tested (about 10 mg/kg bw/day). Abnormalities during parturition and nursing, and effects on growth, were assessed in the various generations.

GLP compliance:

no

Remarks:

predates GLP

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

- M/F ratio per cage: no data in citing sources
- Length of cohabitation: no data in citing sources
- Proof of pregnancy: no data in citing sources
- Further matings after two unsuccessful attempts: no data in citing sources
- After successful mating each pregnant female was caged (how): no data in citing sources
- Any other deviations from standard protocol: no data in citing sources

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 generations

Frequency of treatment:

Presumably daily (given in the diet)

Details on study schedule:

No data in citing sources

Remarks:

Doses / Concentrations:
200 mg/kg diet
Basis:
nominal in diet
equivalent to about 10 mg/kg bw/day for adult rats, and 20 mg/kg bw/day for young rats

No. of animals per sex per dose:

No data in citing sources

Control animals:

yes, plain diet

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen over the course of the study

Remarks on result:

other: Generation: all generations (migrated information)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at dose.

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Conclusions:

Secondary sources report on a pre-GLP four-generation study on DOS, a structurally-related read-across compound for DIDS. Reproduction, suckling and growth were evidently normal in rats of the various generaitons recieving about 10 mg/kg bw/day from the diet (the study NOAEL).

Executive summary:

A pre-GLP four-generation study has been conducted on DOS, a structurally-related read-across compound for DIDS, and is reported in the secondary literature.

 

Rats were treated with a single (relatively low) dose level of 200 mg/kg diet (about 10 mg/kg bw/day) over four generations [no further details in citing sources; screening developmental/reproductive limit tests use a dose level of at least 1000 mg/kg bw/day].

 

Reproduction was considered normal, and no abnormalities were found during parturition or nursing. Growth was evidently normal in each generation. The no-observed-adverse-effect level for fertility effects can therefore be considered about 10 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

No standard data are available on DIDS, and only limited data are available on a structurally-related read-across compound (DOS).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral

No human or laboratory animal data are currently available on the reproductive toxicity potential of DIDS.

The potential for DIDS to bind the oestrogen receptor has been assessed; a negative result was expected in the relative oestrogen receptor binding assay (Danish EPA DB) (OECD, 2012).

Relevant read-across data:

In a limited four-generation study, briefly reported in the secondary literature, normal reproduction, nursing and growth were seen in the various generations of rats fed DOS at 200 mg/kg diet. The NOAEL was about 10 mg/kg bw/day for reproductive toxicity (the only tested dose) (Le Breton, 1952-7).

 

Dermal and inhalation

No human or laboratory animal data are available on the reproductive toxicity of DIDS, or its structurally-related read-across compounds DEHS/DOS or DIDA, resulting from dermal or inhalation treatment.

Short description of key information:
No standard human or laboratory animal data were available on the reproductive toxicity potential of DIDS.

A limited four-generation study on DOS, a structurally-related read-across compound, found no effects on the fertility, nursing or growth of rats given about 10 mg/kg bw/day (Le Breton, 1952-7).

Justification for selection of Effect on fertility via oral route:
Limited four-generation study on a read-across compound.

Effects on developmental toxicity

Description of key information

 DIDS, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day, induced slight maternal toxicity, with an initial body weight loss, reduced food consumption and clinical signs of piloerection. The Mid dose of 300 mg/kg bw/day caused minimal signs of toxicity, with a less pronounced effect on body weight, but there was evidence of slight maternal toxicity. No embryotoxicity was observed in the study based on overall development. Lower mean foetal body weight and an increased number of runts and affected litters indicated that foetotoxicity seen in animals in the High dose group should be considered as secondary to maternal toxicity. There were no malformations attributed to the test item at any dose level. Slight differences in the incidence of retarded ossification in the foetuses of animals in the High dose group were considered to be within the normal range, but may have been related to maternal toxicity. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2019 to August 2019 (end of experiment), final report April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

foetal gender determination via anogenital appearance rather than anogenital distance; method of euthanasia different from method in study plan; temperature (21.2-26.2C) fell outside expected range. No deviations adversely affect integrity of the study

GLP compliance:

yes

Specific details on test material used for the study:

Batch/lot number: ES18-02097
Description: Colourless liquid
Purity: 99.6%
Expiry date: 17 July 2020
Storage conditions: Controlled room temperature (15-25 C, <70% relative humidity)
Vehicle:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Age of animals: Young adult female rats, nulliparous and non-pregnant,
13 weeks old at mating.
Starting body weight: 194 - 258 g (the variation did not exceed ± 20% of the
mean weight)
Acclimation period: at least 14 days

Animal health: Only healthy animals were used for the test, as certified
by the staff Veterinarian.
Cage type: Type II polycarbonate cages were used during mating
and gestation period and Type III polycarbonate cages
were used during the acclimatisation period.
Bedding: SAFE Hygienic Animal Bedding (Batch number:
03027181126, Expiry date 26 November 2021),
produced by J. Rettenmaier & Söhne GmbH+Co.KG
(Holzmühle 1, D-73494 Rosenberg, Germany was used
in the study. Details of bedding quality are reported in
Appendix 8.
Nesting: Arbocel crinklets natural nesting material produced by
J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1,
D-73494 Rosenberg, Germany, Batch number:
05072181025, Expiry date: 12 December 2021) and
SAFE crinklets natural nesting material produced by J.
Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-
73494 Rosenberg, Germany) were used in the study
(Batch number: 05072190726, Expiry date: 26 July
2022). Details of nesting quality are reported in
Appendix 8.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2-26.2°C (target: 22 ± 3°C)
Relative humidity: 34-69% (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Successfully mated animals were housed individually.
Deep wood sawdust was use as bedding to allow digging
and other normal rodent activities. Nest building
material and hiding tunnels were also added to the cages.
The temperature and relative humidity were monitored continuously and recorded twice
on each day during the study. The bedding and nest building material were considered
not to contain any contaminants that could reasonably be expected to affect the purpose
or integrity of the study.

Diet and water supply-
The animals were provided with ssniff® SM Autoclavable Complete Diet for Rats/Mice
– Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
and tap water (in water bottles) as for human consumption, ad libitum. The diet and
drinking water were routinely analysed and are considered not to contain any
contaminants that could reasonably be expected to affect the purpose or integrity of the
study.

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80 in Propylene glycol

Details on exposure:

A constant volume of 5 mL/kg body weight was administered to all dose groups,
including the controls. The individual volume of the treatment was based on the most
recent individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive
(assumed pregnant) female rats daily by oral gavage on a 7-days/week basis,
approximately at similar times, from gestation day 6 (GD 6) to gestation day 19
(GD 19).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed at the Laboratory of Charles River Laboratories Hungary Kft. using a validated HPLC-UV method. Top, middle and bottom samples were taken from the test item formulations two times during the study (during the first week and on the last week of treatment).
Samples were taken in duplicate, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Acceptance criterion of the concentration analysis was set according to the analytical method validation, expected to be at 100 ± 15% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) to be less than 10%.

Details on mating procedure:

The oestrus cycle of female animals was examined a day before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the
morning for approximately 2-3 hours (1 male : 1 female) until 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Duration of treatment / exposure:

Treatment was administered from gestation days 6 to 19

Frequency of treatment:

Treatment was administered on a 7-days a week basis

Duration of test:

Start of experiment: 25 July 2019
End of experiment: 23 August 2019

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 mated females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were set by the Study Director in consultation with the Sponsor, based on the available data and information from previous experimental work, including the results of a 28-Day Oral (gavage) Dose Range Finding Toxicity Study in Wistar Rats (Charles River Laboratories Hungary study code: 18/296-101PE). In that study, no adverse effect was observed in the test item treated group (1000 mg/kg bw/day). Increase of the liver and kidney weights in the test item treated group were recorded, with no correlating adverse liver histopathology findings.
Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose
respectively). The aim is to use the highest dose of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose
level. The oral route was selected since it is a route of administration recommended by the OECD guideline No. 414 and it was considered suitable to provide the exposure
required for this developmental toxicology study.

Maternal examinations:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
Cage-side (general) clinical observations were made twice daily (at the beginning and end of each working day). Only one general clinical observation was made on the first
day (in the afternoon), on the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observation (detailed) was made only
once on necropsy days (in the morning). Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma [4].
On GD 13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intra-uterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8,10, 12, 14, 16, 18 and 20.
Food was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.
Food consumption was also calculated for each interval, including GD 0-6, GD6-10, GD 6-20 and GD 0-20.

No histopathology evaluation was performed in the study (as it was not required for interpretation).

Ovaries and uterine content:

Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital (Euthanimal 40%, 400 mg/mL sodium pentobarbital
solution; Supplier: Alfasan Nederland BV, Batch number: 1811347-03, Expiry date:
31 December 2021) administered by intraperitoneal injection and followed by exsanguination was used for euthanasia.
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; all the gross findings were retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation.
Furthermore, the livers of the dams were weighed and retained in 10% buffered formalin solution for any possible histological examination.
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal
deaths and for the number of live foetuses.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses
was determined according to the appearance of the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination.
For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a
dissecting microscope. The rest of the body was fixed in Sannomiya mixture (a mixture of 920 mL concentrated isopropanol, 30 g sulfosalicylic acid, and 50 mL acetic acid),
then, after fixation, the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcianblue- acetic acid – isopropanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting
microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; photographic records were made additionally.

No histopathology evaluation was performed in the study (as it was not required for interpretation).

Statistics:

Data were collected using the software PROVANTIS v.9 or recorded on appropriate forms from the relevant SOPs of Charles River Laboratories Hungary Kft. (foetal evaluation data) and tabulated with PROVANTIS v.9/Microsoft Office Excel 2010. Statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9 or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) for data tabulated in Excel by an appropriate statistical method. For the SAS v9.2 software package (within the validated Provantis system) the normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of intergroup differences. If either of the Shapiro-Wilk or Levene tests showed significance a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test.
For the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test assessed the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis ANOVA was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi squared test was used for comparing group incidences against the controls.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Thin fur was observed in 2 out of 24 animals in the Control group, in 3 out of 24 animals in the Low dose group, and in 1 out of 23 animals in the High dose group. These changes were most probably incidental, and not considered to be test item related effects.
Piloerection was present in 4 out of 22 animals in the Mid dose group, and 19 out of 23 animals in the High dose group. The finding appeared one day after the start of the treatment (Day7), and peaked for both dose groups at Day 10, the fifth treatment day.
While the distribution of the symptoms indicates that the animals adapted to the treatment, these findings correlate with the observed food intake and body weight loss during the peak period (first week of treatment). Noisy respiration was observed only in the Low dose group (1 out of 24 animals).

Based on the relatively high proportion of this otherwise non-specific finding, piloerection is considered to be a test item related, maternal effect. It is not known if the effect was a systemic effect, or a digestive or other non-systemic cause.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test item related body weight decrease was observed after the onset of treatment in animals in the High dose group.

Subsequently, significantly lower (p < 0.01) body weight gain was observed in animals in the High dose group from Day 6 (the first day of treatment) up to Day 12, and the whole treatment and experimental period. Furthermore, significantly lower corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in the High dose group.
A similar, more transient change, not reaching statistical significance, was observed in animals in the Mid dose group.

These changes were considered to be test item related, adverse effects.

No test item related effect on body weight or body weight parameters were observed in animals in the Low dose group when compared to control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A transient, test item related reduction of the food consumption was observed at the start of the treatment in the test item treated dose groups, reaching statistical significance (p < 0.01) in the periods between Day 6 (the first day of treatment) and Day 12 in the Mid and High dose groups and, while the reduction was compensated during the last week of the treatment, a statistically significant decrease was present when the whole treatment or experimental period was considered, both in the Mid (p < 0.05) and the High (p < 0.01) dose groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effect on gravid uterine weight, or liver weight

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related macroscopic findings were present at necropsy in any of the animals in the study.
Thin fur, correlating with the clinical signs, was observed in one animal in the Low and the High dose group, respectively, but it was considered to be an incidental finding, not a test item related change.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effect on implantation loss

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Description (incidence and severity):

No effect on resorption.

Dead fetuses:

no effects observed

Description (incidence and severity):

No effect on intra-uterine mortality

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Ninety-six females (24 each for the Control, the Low, Mid, and High dose group, respectively) were mated in the study. The number of confirmed pregnant, evaluated dams was 24 in the Control and the Low dose group, 22 in the Mid dose group, and 23 in the High dose group.

Other effects:

no effects observed

Description (incidence and severity):

Corpora lutea and implantation sites
There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the control.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly lower body weights/litter were observed in animals in the Mid (p<0.05), and High dose groups (p<0.01).
While the body weight difference reached significance in the Mid and High dose group, the actual values are within (Mid dose group) or slightly below (High dose group) the expected historical control range (mean ± SD, 3.407 ± 0.225), and therefore not considered to be an adverse test item related effect.

Significantly higher number of foetuses with retarded body weight were observed in the High dose group (p<0.05). These test item related changes were considered to be a secondary effect to the observed maternal toxicity.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Significantly lower body weights/litter were observed in animals in the Mid (p<0.05), and High dose groups (p<0.01).
While the body weight difference reached significance in the Mid and High dose group, the actual values are within (Mid dose group) or slightly below (High dose group) the expected historical control range (mean ± SD, 3.407 ± 0.225), and therefore not considered to be an adverse test item related effect.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no external varieties or malformations in the test item treated groups.
The malformation observed in the Control group (Absent mandible) was considered to be an incidental finding, not related to the treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The observed skeletal abnormalities are summarized in Table 9. Most of the skeletal findings correspond with the current historical control (HC) or the concurrent study control data, or were considered to be incidental findings without dose response (as shown in Table 10). In some cases, skeletal variations (without toxicological significance) shows higher incidence in the test item treated groups, indicating a secondary effect due to maternal toxicity.

Based on the skeletal findings the number of malformed / intact foetuses were comparable with the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups, reaching significance in the Mid and High dose groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

All of the visceral findings (variations) were consistent in general nature and incidence with the concurrent study control data / existing historical control data or showed incidental occurrence, therefore considered as incidental findings. Based on the visceral findings, the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

not specified

Group no./ Designation	Dose Level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg)	Number of mated females	Evaluation No.
1/ Control	0	0	5	24	1501-1524
2/ Low dose	100	20		24	2501-2524
3/ Mid dose	300	60		24	3501-3524
4/ High dose	1000	200		24	4501-4524

Table 1 - Experimental design

 

Table 2 – Summary of the dose formulation analysis

Formulation	30 July 2019		19 August 2019
	Mean measured concentration (mg/mL)	Relative to the nominal concentration (%)	Measured concentration (mg/mL)	Relative to the nominal concentration (%)
Control	Not detected	-	Not detected	-
20 mg/mL	19.5 ± 0.56	97	19.9 ± 0.43	99
60 mg/mL	57.8 ± 0.91	96	60.3 ± 0.36	100
200 mg/mL	199 ± 3.7	100	206 ± 4.12	103

Note: Samples collected freshly on the days indicated in the header of the table.

 

Table 3 – selected body weight parameters

Parameters	Historical control	Dose (mg/kg bw/day)
		0	100	300	1000
Number of evaluated dams	507	24	24	22	23
Body weight on GD20 (g)	316.4±23.1	324.2	321.9	319.0	307.0#	DN
Body weight gain GD6-20 (g)	83.1±14.1	82.1	81.8	78.3	63.8##	DN
Body weight gain GD0-20 (g)	104.3±16.2	104.2	102.2	97.5	84.5##	DN
Corrected body weight on GD20 (g)	258.2±18.7	267.0	265.5	263.9	251.6#	DN
Corrected body weight gain GD0-20 (g)	46.1±11.8	47.0	45.8	42.3	29.0**	U
Net body weight gain GD6-20 (g)	24.9±9.8	24.9	25.5	23.1	8.3**	U

Notes: Historical control data is mean ± 1SD. Body weight data were rounded to one decimal place. Corrected and net weight/weight gains refer to body weight values minus the weight of the gravid uterus.

U: *=p<0.05; **=p<0.01; Dunn two sided test

DN: #=p<0.05; ##=p<0.01; Dunnett two sided test

 

Table 4 – Summary of pregnancy data

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of mated females	24	24	24	24
Pre-terminal death or euthanasia	0	0	0	0
Number of non-pregnant females	0	0	2	1
Number of evaluated females with≤5 implantation sites	0	0	0	0
Number of evaluated females on GD20 (Caesarean section)	24	24	22	23

 

Table 5 – Summary of the intra-uterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of evaluated dams	24	24	22	23
Mean number of corpora lutea	11.63	11.33	10.46	10.76	NS
Pre-implantation loss, mean	1.67	1.71	1.82	0.91	NS
Pre-implantation loss (%), mean	13.10	14.17	15.18	7.53	NS
Mean number of implantations	9.96	9.63	9.59	10.35	NS
Early embryonic loss, mean	0.21	0.00	0.05	0.13	NS
Early embryonic loss (%), mean	2.27	0.00	0.41	1.16	NS
Late embryonic loss, mean	0.08	0.00	0.14	0.22	NS
Late embryonic loss (%), mean	1.11	0.00	1.17	2.15	NS
Dead foetuses, mean	0.00	0.00	0.00	0.00	NS
Dead foetuses (%), mean	0.00	0.00	0.00	0.00	NS
Post-implantation loss, mean	0.29	0.00	0.18	0.35	NS
Post-implantation loss (%), mean	3.38	0.00	1.58	3.31	NS
Total intra-uterine mortality, mean	1.96	1.71	2.00	1.26	NS
Total intra-uterine mortality (%), mean	16.28	14.17	16.73	10.56	NS
Viable foetuses, mean	9.67	9.63	9.41	10.00	NS

Notes: Most important parameters are shown in bold.

NS: Statistically not significant when compared to the vehicle control.

NA: Not applicable

 

Table 6 – Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	100	300	1000
Number of examined litters	24	24	22	23
Viable foetuses, mean	9.67	9.63	9.41	10.00	NS
Male foetuses, mean	4.67	4.75	5.05	5.04	NS
Female foetuses, mean	5.00	4.88	4.36	4.96	NS
Total number of foetuses	232	231**	207	230	CH
Total number of male foetuses	112	114	111	116	NS
Total number of female foetuses	120	117	96	114	NS
Sex distribution (% of males/females)	48/52	49/51	54/46	50/50	NS
Mean foetal weight/litter (g)	3.435	3.404	3.367*	3.135**	U
Number of foetuses with retarded body weight	9	6	10	58**	CH
Number of affected litters (with runts)	7	4	7	14*	CH

 

Table 7 – Summary table of the external abnormalities

Parameter
	0	100	300	1000
Total number of examined litters	24	24	22	23
Total number of examined foetuses	118	114	104	113
Total number of intact (normal) foetuses	115	112	103	113
Total number of foetuses / litters with malformation	0 / 0	0 / 0	0 / 0	0 / 0
Total number of foetuses / litters with variation	3 / 3	2 / 2	1 / 1	7 / 7

Notes: Numbers represent the number of abnormalities / number of affected litters

 

Table 9 – Details of the visceral abnormalities

Parameter			Dose (mg/kg bw/day_				HC data
			0	100	300	1000
Total number of examined litters			24	24	22	23	507
Total number of examined foetuses			118	114	104	113	2608
Visceral malformations
Visceral variations
Brachiocephalic trunk, short	Litter incidence	n	0	0	0	1	23
		%	0.0	0.0	0.0	4.3	4.5
	Foetal incidence	n	0	0	0	1	24
		%	0.000	0.000	0.000	0.885	0.920
Liver lobe, fused	Litter incidence	n	0	1	0	0	--
		%	0.0	4.2	0.0	0.0	--
	Foetal incidence	n	0	1	0	0	--
		%	0.000	0.877	0.000	0.000	--
Renal papilla, small	Litter incidence	n	1	0	0	1	16
		%	4.2	0.0	0.0	4.3	3.2
	Foetal incidence	n	1	0	0	1	17
		%	0.847	0.000	0.000	0.885	0.652
Ureter convoluted	Litter incidence	n	1	0	0	0	7
		%	4.2	0.0	0.0	0.0	1.4
	Foetal incidence	n	1	0	0	0	7
		%	0.847	0.000	0.000	0.000	0.268
Thymic cord	Litter incidence	n	2	1	1	5	51
		%	8.3	4.2	4.5	21.7	10.1
	Foetal incidence	n	2	1	1	5	60
		%	1.695	0.877	0.962	4.425	2.31

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted when compared to the control group.

--: No data

 

Table 10 – summary table of the skeletal abnormalities

Parameter	Dose (mg/kg bw/day)
	0	100	300	1000
Total number of examined litters	24	24	22	23
Total number of examined foetuses	114	117	103	117
Total number of intact (normal) foetuses	95	93	71^CH*	64^CH**
Total number of foetuses / litters with malformation	4 / 4	2 / 2	7 / 5	4 / 3
Total number of foetuses / litters with variation	15 / 12	22 / 12	25^CH*/ 12	49^CH**/ 19

Notes: Numbers represent the number of abnormalities / number of affected litters

CH: Chi²test; * = p < 0.05; ** = p < 0.01

 

Table 11 – Details of the skeletal abnormalities

Parameter			Dose (mg/kg bw/day)				HC data
			0	100	300	1000
Total number of examined litters			24	24	22	23	507
Total number of examined foetuses			114	117	103	117	2599
Skeletal malformations
Transverse process fused; Pelvic girdle, malpositioned	Litter incidence	n	2	1	5	2	7
		%	8.3	4.2	22.7	8.7	1.4
	Foetal incidence	n	2	1	7	3	7
		%	1.754	0.855	6.796	2.564	0.269
Multiple malformed vertebrae (hemicentric, hemivertebrae, misshapen)	Litter incidence	n	1	1	0	0	--
		%	4.2	4.2	0.0	0.0	--
	Foetal incidence	n	1	1	0	0	--
		%	0.877	0.855	0.000	0.000	--
Mandible, short, fused	Litter incidence	n	1	0	0	0	1
		%	4.2	0.0	0.0	0.0	0.2
	Foetal incidence	n	1	0	0	0	1
		%	0.877	0.000	0.000	0.000	0.038
Rib, branched	Litter incidence	n	1	0	0	0	--
		%	4.2	0.0	0.0	0.0	--
	Foetal incidence	n	1	0	0	0	--
		%	0.877	0.000	0.000	0.000	--
Humerus short	Litter incidence	n	1	0	0	0	--
		%	4.2	0.0	0.0	0.0	--
	Foetal incidence	n	1	0	0	0	--
		%	0.877	0.000	0.000	0.000	--
Scapula short, bent	Litter incidence	n	1	0	0	1	--
		%	4.2	0.0	0.0	4.3	--
	Foetal incidence	n	1	0	0	1	--
		%	0.877	0.000	0.000	0.855	--
Skull: Incomplete ossification (More than 3)	Litter incidence	n	3	5	9	11	13
		%	12.5	20.8	40.9	47.8	2.6
	Foetal incidence	n	3	10	21^CH**	20^CH**	18
		%	2.632	8.547	20.388	17.094	0.693
Skull: Hyoid Body Unossified	Litter incidence	n	5	6	5	8	3
		%	20.8	25.0	22.7	34.8	0.6
	Foetal incidence	n	5	9	6	16^CH*	5
		%	4.386	7.692	5.825	13.675	0.192
Sternum: Unossified Sternebra (3 or More)	Litter incidence	n	2	1	2	13	--
		%	8.3	4.2	9.1	56.5	--
	Foetal incidence	n	2	3	2	27^CH**	--
		%	1.754	2.564	1.942	23.077	--
Sternum: Misaligned	Litter incidence	n	0	0	2	0	1
		%	0.0	0.0	9.1	0.0	0.2
	Foetal incidence	n	0	0	2	0	1
		%	0.000	0.000	1.942	0.000	0.038
Ribs: Wavy, Extreme	Litter incidence	n	3	0	1	2	132
		%	12.5	0.0	4.5	8.7	26.0
	Foetal incidence	n	3	0	1	3	202
		%	2.632	0.000	0.971	2.564	7.772
Vertebrae: 2 or More Dumbbell or Asymmetric Ossification	Litter incidence	n	2	1	3	1	51
		%	8.3	4.2	13.6	4.3	10.1
	Foetal incidence	n	3	1	3	1	56
		%	2.632	0.855	2.913	0.855	2.155
Vertebrae: Dumbbell Shaped	Litter incidence	n	0	5	3	0	6
		%	0.0	20.8	13.6	0.0	1.2
	Foetal incidence	n	0	6^CH*	3	0	6
		%	0.000	5.128	2.913	0.000	0.231
Vertebrae: Unossified centra or arch	Litter incidence	n	2	2	2	5	--
		%	8.3	8.3	9.1	21.7	--
	Foetal incidence	n	2	4	2	14^CH**	--
		%	1.753	3.419	1.942	11.966	--
Vertebrae: Bipartite Ossification	Litter incidence	n	1	3	1	0	11
		%	4.2	12.5	4.5	0.0	2.2
	Foetal incidence	n	1	3	1	0	11
		%	0.877	2.564	0.971	0.000	0.423
Limbs (C/T): Unossified Carpal Bone (2 or More)	Litter incidence	n	2	1	2	2	--
		%	8.3	4.2	9.1	8.7	--
	Foetal incidence	n	2	3	2	6	--
		%	1.754	2.564	1.942	5.128	--
Limbs (C/T): Unossified Tarsal Bone	Litter incidence	n	1	1	1	5	--
		%	4.2	4.2	4.5	21.7	--
	Foetal incidence	n	1	1	2	12^CH**	--
		%	0.877	0.855	1.942	10.256	--
Limbs (C/T): Unossified Pubis, Ischium	Litter incidence	n	0	1	0	1	--
		%	0.0	4.2	0.0	4.3	--
	Foetal incidence	n	0	1	0	1	--
		%	0.000	0.855	0.000	0.855	--

Notes: Numbers represent the number (n) or ratio (%) of abnormalities.

HC: historical control (data provided where considered useful)

No statistically significant differences were noted compared to the control group

--: No data

CH: Chi²test; * = p <0.05; ** = p < 0.01

Conclusions:

In conclusion, CEREPLAS DIDS, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced a slight maternal toxicity, with an initial body weight loss, reduced food consumption and clinical signs of piloerection. The Mid dose of 300 mg/kg bw/day had minimal signs with a less expressed effect on body weight, but there was evidence of a slight maternal toxicity. No embryotoxicity was observed in the study based on overall development. The lower mean foetal body weight and the increased number of runts and affected litters indicates foetotoxicity seen in animals in the High dose group should be considered as secondary to maternal toxicity. There were no malformations attributed to the test item at any dose level. Slight differences in the incidence of retarded ossification in the foetuses of animals of the High dose group were considered to be within the normal range, but may have been related to maternal toxicity.

Executive summary:

This developmental toxicity study (OECD 414, 2001 version as required by Sponsor) was performed to assess the effects of the test item CEREPLAS DIDS on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day 6 (GD 6) up to and including gestation day 19 (GD 19), where sperm positive day was counted as day 0 of pregnancy (GD 0). Control dams were treated with the vehicle (1% Tween 80 in propylene glycol) only. Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD 20. The dose levels were set by the Study Director in consultation with the Sponsor, based on the available data and information from previous experimental work, including the

results of a 28-Day Oral (gavage) Dose Range Finding Toxicity Study in Wistar Rats

[8].

Based on the results from the Dose Range Finding study, doses of 1000, 300 and 100 mg/kg bw/day were selected for the main study (designated High, Mid and Low dose respectively). The aim is to use the highest dose of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering, and to obtain a NOAEL at the lowest dose level.

 

Test item formulations were analysed for concentration and homogeneity two times during the treatment period using a validated HPLC-UV method. Simultaneously, vehicle control formulations were analysed for the test item. Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external, visceral and skeletal abnormalities. Placentas were examined macroscopically. The number of confirmed pregnant, evaluated dams was 24 in the Control and Low, 22 in the Mid dose group, and 23 in the High dose groups.

 

Results

All test item formulations were within the range of 96-103% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

No mortality was observed in the study.

Piloerection was present in 4 out of 22 animals in the Mid dose group, and 19 out of 23 animals in the High dose group. These adverse effects were considered to be test item related.

Test item related body weight decrease was observed after the onset of treatment in animals in the High dose group. Consequently, significantly lower body weight gain, and lower corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in the High dose group. A similar, more transient change, not reaching statistical significance, was observed in animals in the Mid dose group. No test item related effect on the body weight parameters were observed in animals in the Low dose group when compared to controls. Transient, test item related reduction of the food consumption was observed at the start of the treatment in the test item treated groups, reaching statistical significance in the Mid and High dose group. No test item related macroscopic findings were present at necropsy in any of the animals in the study.

Taking the above maternal findings together, it is considered that were was slight/minimal maternal toxicity present in animals in the Mid dose group, and more significant maternal toxicity in animals in the High dose group. There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls. There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups. Significantly lower foetal body weights were observed in the test item treated groups. The well-expressed reduction in body weight in animals in the High dose group correlated with the increased number of runts and affected litters in this dose group. There was no toxicologically significant difference in the number of runts and affected

litters between the control and the Low and Mid dose groups. There were no test item related effects on external or visceral development of foetuses in the study. The number of skeletal variations was higher in the test item treated groups when compared to the controls, reaching statistical significance in the Mid and High dose group due to maternal toxicity. Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as a test item related effect.

 

In conclusion, CEREPLAS DIDS, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced a slight maternal toxicity, with an initial body weight loss, reduced food consumption and clinical signs of piloerection. The Mid dose of 300 mg/kg bw/day had minimal signs with a less expressed effect on body weight, but there was evidence of a slight maternal toxicity. No embryotoxicity was observed in the study based on overall development. The lower mean foetal body weight and the increased number of runts and affected litters indicates that foetotoxicity seen in animals in the High dose group should be considered as secondary to maternal toxicity. There were no malformations attributed to the test item at any dose level. Slight differences in the incidence of retarded ossification in the foetuses of animals in the High dose group were considered to be within the normal range, but may have been related to maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A standard OECD Guideline 414 study is available on DIDS.

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

Oral

A guideline OECD 414 study has been performed using DIDS as the test substance. No adverse effects were seen in reproduction or development at the highest dose (1000 mg/kg bw/day)

 

Relevant read-across data: 

In a limited four-generation study, briefly reported in the secondary literature, no abnormalities during parturition or nursing and no effects on growth, were seen in the various generations of rats fed DOS at 200 mg/kg diet. The NOAEL was about 10 mg/kg bw/day for both maternal and developmental toxicity (the only tested dose) (Le Breton, 1952-7).

 

Dermal and inhalation

No human or laboratory animal data are available on the developmental toxicity of DIDS, or its structurally-related read-across compounds DOS/DEHS and DIDA, resulting from dermal or inhalation treatment. Instead, the study conducted via the oral route addresses these endpoints.

Justification for selection of Effect on developmental toxicity: via oral route:

OECD Guideline developmental toxicity study on DIDS conducted to GLP

Justification for classification or non-classification

No developmental effects were seen in a standard OECD Guideline 414 study on DIDS. No adverse effects were seen on reproduction or development in a limited four-generation study on a structurally-related read-across compound (DOS). The available data indicate that the classification of DIDS as a reproductive or developmental toxicant under CLP Regulation (EC) 1272/2008 is not warranted.