Rape oil, reaction products with diethanolamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Repeated dose toxicity studies on structural analogues exist which screen for some of the fertility endpoints.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Although no specific reproductive toxicity studies have been conducted with HE Rape Oil, reaction product with diethanolamine, the available repeated dose toxicity (28-day, 90-day and 2-year) conducted on structurally similar substances did not show any effects on reproductive organs at the highest treatment levels. While recognizing that a reproductive/developmental screening study and/or a multigeneration or an extended one generation reproductive study would be able to address critical parameters for reproductive toxicity assessment such as sperm quality and other fertility parameters, according to a recently published paper by Aulmann W (2012) a well conducted ‘full scale’ repeated dose toxicity study (28-day and/or 90-day after 1995) in combination with a developmental toxicity study is considered to provide more robust and more relevant information for the assessment of reproductive toxicity than the information obtained from a screening study. Aulmann bases his assessment on the following key observations:

• Most male reproductive toxicants can be detected with adequate histopathology of the testes. A compound without adverse effect on reproductive organ weights or on testes histopathology especially at dose levels producing significant toxicity in other organ systems would very likely not be detected as a male reproductive toxicity in OECD 421/422 (Screening studies);

• A repeated dose toxicity (RDT) study including histopathology of the female reproductive organs and a continuous observation of oestrous cyclicity is also suitable to detect female reproductive toxicant. However, it has also been shown that RDT are less sensitive than female fertility studies. This issue has mostly been related to fixation and staining methods. Nevertheless RDT studies may be used for a screening of potential impairment of fertility, especially with improved fixation and staining methods;

• RDT are able to detect the majority of compounds affecting fertility with many different modes of action including endocrine modulation; consequently an adequately designed RDT study is expected to provide comparable information on potential effects on fertility as an OECD 421/422;

• One might fail to see effects on pup viability or impaired parturition – in general these effects are considered rare events.

In addition to the discussion, Aulmann provided an overview of examinations required in a ‘well conducted’ repeated dose toxicity study to support the above conclusions. Therefore based on a review of all existing repeated dose toxicity data to assess reproductive toxicity of HE Rape Oil, reaction product with diethanolamine against the set criteria by Aulmann, it has been concluded that the NTP studies contained all examinations that are critical to the reproductive toxicity assessment outlined by Aulmann and others. The subchronic NTP study also included sperm quality as well as vaginal cytology and estrous cycle examinations. However the key deficiencies were:

• Fixation and staining method;

• Dermal as opposed to oral study suggesting lower systemic exposure; however, dermal penetration studies in rats reveal 30% bio-availability suggesting ‘sufficient exposure’ of about 100 mg/kg/day in 14 week study

Therefore under conditions of normal and foreseeable use HE Rape Oil, reaction product with diethanolamine is not expected to have any effects on fertility and therefore this endpoint has been waived on the basis of animal welfare and on the available repeated dose toxicity and developmental toxicity studies.

Short description of key information:
Data waived since scientifically unjustified. Data waived since data from sub-chronic and chronic repeated dose toxicity studies conducted on structural analogues did not reveal any adverse effects on reproductive organs up to the highest tested doses.

Justification for selection of Effect on fertility via oral route:
Although no specific reproductive toxicity studies have been conducted testing for reproductive toxicity is not warranted. This is due to the fact that studies investigating the repeated dose toxicity of structurally similar substances following 28-d, 90-d or 2-yr chronic exposure did not show any effects on reproductive organs at the highest treatment levels. Hence, under conditions of normal and foreseeable use the substance is not expected to have any effects on fertility.

Effects on developmental toxicity

Description of key information

Structurally similar substance amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) was not toxic to pregnant rats and did not reveal any embryotoxic or teratogenic potential at dose levels up to 1,000 mg/kg/d. Therefore, based on the read across study HE Rape Oil, reaction product with diethanolamine is not considered to be a developmental toxicant. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read across study conducted with the structural analogue amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was conducted according to the OECD Guideline 414 (Teratogenicity) and the EU Directives 87/302/EEC and 91/325/EEC. Compliance to GLP has been claimed through the GLP regulations according to the Chemical Act (FRG), March 14, 1990.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 133, May 30, 1988; 87/302/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 180, March 01, 1991; 91/325/EEC

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wiga D-Sulzfeld
- Age at study initiation: 8-10 wk
- Weight at study initiation: 209 g (mean)
- Housing: Single animal in Makrolon Type M3 cage (Ebeco) with standard softwood bedding
- Diet: Pelleted Altromin Maintenance Diet 1324, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 43-66
- Air changes (per h): 10-15
- Photoperiod (h dark/h light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Arachidis oil, DAB 9

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test material was suspended in Arachidis oil, DAB 9 such that the required dose per kg body weight was contained in 5 mL.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant

Duration of treatment / exposure:

From Day 6 up to Day 15 post coitum

Frequency of treatment:

Once daily

Duration of test:

20 d

Remarks:

Doses / Concentrations:
0, 100, 300 and 1,000 mg/kg/d
Basis:
actual ingested
(Dosing on the basis of body weight of Day 6 post coitum)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of toxicological examinations done before (Report No. 486 = TBD 830034, June 27, 1983) (details not reported)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examination: On Day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: All maternal organs, with emphasis on the uterus and uterine contents

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
- Other: Position of fetus in the uterus

Fetal examinations:

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

(See Table 1 for exact number of fetuses examined)

Statistics:

The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the
comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distribution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Indices:

- Pre-implantation loss (%) = [(Number of corpora lutea - number of implantations)/number of corpora lutea] X 100
- Post-implantation loss (%) = [(Number of implantations - number of live fetuses)/number of implantations] X 100
- Sex ratio (%) = [(number of males/females)/number of fetuses] X 100

Historical control data:

None

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality: No mortality at any dose level.

Clinical symptoms: Salivation and propulsion of the head in all dose groups. Additionally, the highest dose group showed a severe salivation. These symptoms were noted variable in the individual groups during the application period.

Body weight: No treatment-related effects on body weight gain were observed in the dams.

Necropsy: No macroscopic changes were observed in the survived dams except for one dam at 100 mg/kg/d, which showed greenish-brownish fluid in the uterine horn.

Placenta and uterus weight: No significant differences between the control and the treatment groups.

Reproduction data: Pre-implantation loss was not affected by the treatment. The post-implantation loss and total embryonic deaths were significantly increased in all treatment groups. However, these findings were considered to be incidental because the values in the 100 mg/kg/d group were significantly greater than other two higher dose groups and in each group there was one single female with a high incidence of embryonic death.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Body weight: The weights of live fetuses exhibited no significant differences on a litter and individual basis.

Sex ratios: The sex ratio of the fetuses was not affected by the treatment.

External examinations: No macroscopic findings were observed at external examination of fetuses which were considered to be an effect of the treatment. 1 dead fetus in control and 7 dead fetuses (4 out of 7 partly mummified) in 100 mg/kg/d group were recorded. One fetus showed a stump tail at 300 mg/kg/d and paleness was observed in one fetus at 1,000 mg/kg/d. These singular findings are normal observations in the animal strain used.

Visceral examination: No treatment-related abnormalities.

Skeletal examination:
(i) Retardations: No significant finding at 100 mg/kg/d. Two sternebrae were non-ossified in 19 and 29 fetuses (statistically significant) at 300 and 1,000 mg/kg/d, respectively. Statistically significant increase in the number of fetuses with incomplete ossification of skull bones (17 fetuses) and decrease in the number of fetuses with incomplete ossification of 13th rib (0 fetus) was observed at 1,000 mg/kg/d. The increased "incomplete ossified skull bones" was essentially due to only 2 dams. The other statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation and were within the normal range of variation for this strain.
(ii) Variations: No variations in any group.
(iii) Malformations: One fetus with stump tail and missing vertebrae coccigycae at 300 mg/kg/d (not considered to be treatment-related).

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 2. Summary of performance of mated females

Treatment dose (mg/kg/d)	0	100	300	1,000
No. of mated females	30	30	30	30
No. of pregnant females	30	29	28	29
No. of females with premature litter	1	0	2	3
No. of mortalities	0	0	0	0
No. of females with live fetuses at termination	29	29*	26	26

* One dam out of these was not included because the weights of fetuses were not determined

Conclusions:

Based on the results, the structurally similar amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) was not found to be cumulative toxic to pregnant rats and did not reveal any embryotoxic or teratogenic potential at dose levels up to 1,000 mg/kg/d.

Executive summary:

A study was carried out to assess the effects of structurally similar amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) on

embryonic and foetal development in pregnant Sprague-Dawley CD rats following the procedures indicated by the OECD guideline 414 (Teratogenicity) and the EU Directives 87/302/EEC and 91/325/EEC.

The test material was administered to groups of 30 female rats orally by gavage at dose levels of 0, 100, 300 and 1,000 mg/kg/d, once daily from Day 6 to day 15 of gestation inclusive. Control animals were dosed with the vehicle alone (arachidis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on Day 0, 6, 16 and 20 of gestation. All surviving females were sacrificed on Day 20 of gestation and the foetuses were removed by caesarean section. At necropsy the females were examined macroscopically. Live foetuses were weighed, sexed and were examined for visceral and skeletal abnormalities.

No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all treatment groups were salivation and propulsion of the head. Additionally, the highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg/day group (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The figures of post-implantation loss, embryonic deaths and total foetuses showed some deviations which were considered to be non-treatment-related. Mean foetal placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus in 300 mg/kg/d group showed a stump tail and missing vertebrae coccigycae. Further, the figures of skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related.

Based on the results the test substance was found not to be cumulative toxic to pregnant rats and did not reveal any embryotoxic or teratogenic potential to Sprague-Dawley CD rats at dose levels up to 1,000 mg/kg bw/day.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The information requirement for this tonnage band is sufficiently met with the available data.

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

A study was carried out to assess the effects of structurally similar substance amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) on embryonic and foetal development in pregnant Sprague-Dawley CD rats following the procedures indicated by the OECD guideline 414 (Teratogenicity) and the EU Directives 87/302/EEC and 91/325/EEC. The test substance was administered to groups of 30 female rats orally by gavage at dose levels of 0, 100, 300 and 1,000 mg/kg/d, once daily from Day 6 to Day 15 of gestation inclusive. Control animals were dosed with the vehicle alone (arachidis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on Day 0, 6, 16 and 20 of gestation. All surviving females were sacrificed on Day 20 of gestation and the foetuses were removed by caesarean section. At necropsy the females were examined macroscopically. Live foetuses were weighed, sexed and were examined for visceral and skeletal abnormalities.

No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all treatment groups were salivation and propulsion of the head. Additionally, the highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg/day group (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The figures of post-implantation loss, embryonic deaths and total foetuses showed some deviations which were considered to be non-treatment-related. Mean foetal placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus in 300 mg/kg/d group showed a stump tail and missing vertebrae coccigycae. Further, the figures of skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Based on the results, the structural analogue amides, C12-18(even-numbered) and C18(unsatd.), N,N-bis(hydroxyethyl) was found not to be cumulatively toxic to pregnant rats and did not reveal any embryotoxic or teratogenic potential to Sprague-Dawley CD rats at dose levels up to 1,000 mg/kg bw/d (Pittermann W, 1994).

Justification for selection of Effect on developmental toxicity: via oral route:
OECD 414 GLP compliant study available on structurally similar substance.

Justification for classification or non-classification

The available data conducted with structural analogues suggests that HE Rape Oil, reaction product with diethanolamine is not a reproductive toxicant with regard to fertility or developmental effects. Therefore no classification is required according to EC criteria (67/548/EEC) and CLP criteria (EC 1272/2008).