A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data provided by the ECHA on request of Cytec Surface Specialties NV/SA. Although details on the results are lacking, the information has been reviewed by ECHA, and is considered reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V

Deviations:

not specified

GLP compliance:

yes

Species:

rat

Strain:

other: Wistar Hoe: WISKf (SPF 71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: unknown
- Age at study initiation: unknown
- Weight at study initiation: unknown
- Fasting period before study: unknown
- Housing: unknown
- Diet (e.g. ad libitum): unknown
- Water (e.g. ad libitum): unknown
- Acclimation period: unknown


ENVIRONMENTAL CONDITIONS
- Temperature (°C): unknown
- Humidity (%): unknown
- Air changes (per hr): unknown
- Photoperiod (hrs dark / hrs light): unknown

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: unknown


DIET PREPARATION
- Rate of preparation of diet (frequency): unknown
- Mixing appropriate amounts with (Type of food): unknown
- Storage temperature of food: unknown


VEHICLE
- Justification for use and choice of vehicle (if other than water): unknown
- Concentration in vehicle: unknown
- Amount of vehicle (if gavage): unknown
- Lot/batch no. (if required): unknown
- Purity: unknown

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Remarks:

Doses / Concentrations:
62.5 mg/bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
250 mg/bw/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1000 mg/bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes

Details on study design:

no data

Positive control:

no data

Observations and examinations performed and frequency:

no data

Sacrifice and pathology:

no data

Other examinations:

no data

Statistics:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No deaths were observed. Signs of toxicity in high and intermediated dosed animals included increased salivation from days 8 and 11 respectively in both sexes with in addition from day 22 onwards, irregular respiration, stilted gait and squatting posture in high dosed females. No signs of toxicity were observed in low dose animals.

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weights.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment related hematological or clinical chemistry effects were observed and no effects were observed in urine.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effects were observed for organ weights.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic abnormalities were seen in the livers of 2 high dose females and consisted of swelling in one and rounding of borders in second.
Microscopic changes were only observed at the border between the glandular and forestomach and consisted of slight hyperplasia (including hyperkeratosis and slightly increased inflammatory reactions).
These changes were observed in 3 male and 2 female high dose rats.
No changes were observed at lower doses.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects were observed up to the high dose level

Key result

Critical effects observed:

no

The results on repeated dose toxicity provided in the IUCLID5 of ECHA were as follows:

NOAEL = 62.5 mg/kg bw/d (nominal) Original NCD unit is mg/kg/day

NOEL = 62.5 mg/kg bw/d (nominal) Original NCD unit is mg/kg/day

Based on the limited information on the results, these values most probably are based on clinical observations at and above 250 mg/kg bw/day.

For the present dossier, in addition to the limited information on repeated dose toxicity as provided by ECHA, also a recently performed combined repeated dose and reproductive/developmental toxicity screening study (OECD 421) is available. In this OECD 421 study, similar clinical observations were observed which were considered to be due to the taste/irritancy of the test substance and therefore not toxicologically relevant. The systemic NOAEL for this study is set at 1000 mg/kg bw/day. 

The information as provided by the ECHA stated effects in organs:

• Macroscopic abnormalities were seen in the livers of 2 high dose females and consisted of swelling in one and rounding of borders in second. However, at microscopic observations, no abnormalities were recorded. It is anticipated that in line the observations in the oral reproductive/developmental screening test in rats these effects on the liver are considered to represent an adaptive response, and are not considered to be adverse or toxicologically relevant.
• The reported microscopic changes in the information provided by ECHA were only observed at the border between the grendular and forestomach and consisted of slight hyperplasia (including hyperkeratosis and slightly increased inflammatory reactions). These microscopic changes were observed in high dose animals only (1000 mg/kg bw/day). These observations are in line with the observations in the oral reproductive/developmental screening test in rats and are most likely due to the irritancy effect of the test article.
• The local NOAEL is therefore set at 250 mg/kg bw/d, which is in line with the observations in both studies. These local effects are most likely caused by the gavage and irritancy effect of the test article and are not considered relevant for human risk assessment.

Conclusions:

Under the study conditions, no systemic effects were observed up to and including 1000 mg/kg bw/day. Based on the effects observed in the 28-day repeated dose toxicity study on rats, the NOAEL value for local effects was determined at 250 mg/kg b.w and the NOAEL for systemic effects at 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the short-term repeat dose toxicity of the test substance in rats. Wistar Hoe: WISKf (SPF 71) rats were used in this study. The test substance was administered by oral gavage to male and female rats at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d. The treatment revealed local toxicity in high dose males and females, characterized by macroscopic and microscopic changes in the stomach suggestive of local irritating effects of repeat dose gavage administration. Under the study conditions, no systemic effects were observed in rats up to and including 1000 mg/kg bw/d and the systemic toxicity NOAEL was determined to be 1000 mg/kg bw/d (Anonymous, 1993).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 30, 2010 to January 31, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 421: Reproduction/Developmental Toxicity Screening Study

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

(specific gravity 1.125)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared on a weekly basis and were homogenised to a visually acceptable level on a daily basis. Adjustment was made for specific gravity of the vehicle (1.125) and test substance (1.14).
Storage conditions: In a refrigerator protected from light.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted once prior to start of the study and weekly (each formulation) during the treatment phase, according to a validated method. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-44, 53 and 56 days).

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

not needed

Observations and examinations performed and frequency:

Mortality / Viability:
At least twice daily. The circumstances of any death were recorded in detail.

Clinical signs:
At least once daily from start of treatment onwards. The time of onset, grade and duration of any observed signs was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Body weights:
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and
20 post-coitum and during lactation on Days 1 and 4.

Food consumption:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of
mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

Water consumption:
Subjective appraisal was maintained during the study period. No treatment related effect was suspected.

Sacrifice and pathology:

Animals surviving to scheduled necropsy were deeply anaesthetized using an isoflurane combination and subsequently exsanguinated.

Necropsy were conducted on the following days:
Females which deliver: Lactation Days 5-7.
Females which fail to deliver: Post-coitum Day 27 (females with evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Spontaneous deaths (Female no. 76): As soon as possible after death and always within 24 hours. Recognizable foetuses were examined externally, sexed (if possible), and euthanized by decapitation (if necessary)

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant clinical signs noted up to 1000 mg/kg.
Salivation was noted for animals of all treated groups, however, this was considered to be due to the taste/irritancy of the test substance, and was not considered to be toxicologically relevant. Salivation was also noted on one occasion for a single control female; this was incidental in nature.
Other incidental findings included rales, which was seen for one animal each at 100, 300, and 1000 mg/kg, and piloerection that was seen for one female each at 300 and 1000 mg/kg, and scabs on the cheek. Lethargy, flat posture and ptosis were seen for one female at 100 mg/kg, however, due to the slight nature of the effects and their limited incidence, these signs were also considered to be incidental in nature, and thus not toxicologically relevant.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death, with female (1000 mg/kg) found dead on day 19 of the treatment period. At macroscopic examination, this female was found with beginning autolysis and greenish fluid in the uterus. The cause of mortality could not be determined.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on body weights or body weight gains were seen with treatment up to 1000 mg/kg.
At 1000 mg/kg, body weight gains were significantly higher for females from Days 4-7 and Day 17 of the post coitum period. This was not considered to be toxicologically relevant because the differences from controls were relatively slight and reduced body weight gain or weight loss would be expected if treatment related toxicity were evident.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on absolute or relative food consumption were seen with the test substance treatment up to 1000 mg/kg.
Absolute food consumption was significantly higher for females over Days 1-8 of the premating period at 100 and 1000 mg/kg, and relative food consumption was significantly higher for females over Days 1-8 of the premating period at 300 and 1000 mg/kg, respectively. Males at 1000 mg/kg also had higher food consumption over Days 8-15 of the premating period. The increases compared to control values were only very slight, and were thus not considered to be toxicologically relevant.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights and organ to body weight ratios were unaffected by treatment up to 1000 mg/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related findings were noted in the stomach of males. At 300 mg/kg and 1000 mg/kg, the number of animals with a thickened limiting ridge of the stomach noted at necropsy was significantly higher than controls. There were 5/10 and 7/10 males noted with this in the 300 and 1000 mg/kg groups, respectively.
Two males at 300 mg/kg were noted with reddish foci on the stomach glandular mucosa, and a single male at 1000 mg/kg was noted with irregular surface of the limiting ridge of the stomach. Because a significant increase in stomach findings for males in these groups were seen, a relationship to treatment cannot be excluded, despite the limited incidence seen.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic findings were noted in the stomach of males of Groups 3 and 4 and in the liver of males of Group 4.
Stomach:
- Hyperplasia of the forestomach / limiting ridge was noted in 5/10 males of Group 3 (minimal) and 5/10 males of Group 4 (4/10: minimal, 1/10: slight). This was also seen in 1/10 males of Groups 1 and 2 (100 mg/kg) at a minimal degree
- Lymphogranulocytic inflammation of the forestomach and/or limiting ridge was recorded at minimal degree in 4/10 Group 4 males. Minimal lymphogranulocytic inflammation of the stomach in the other Groups ranged from 1/10 in Groups 1 and 2 and 2/10 in Group 3. In these groups the inflammation was only located around the limiting ridge.
In females there were 2/10 Group 4 rats with a minimal degree of hyperplasia of the forestomach compared to 1/10 in Group 1. Since there were no gross lesions in the female stomach, this very minimal increase in incidence of hyperplasia of the forestomach in females was considered to be within background ranges, and thus not toxicologically relevant.
Liver:
- Hepatocellular hypertrophy was noted at minimal degree in 2/10 Group 4 males. While this constitutes a treatment related finding, it most likely represents an adaptive response and thus was not considered to be adverse or toxicologically relevant.
The remainder of the recorded microscopic findings were within the normal range of background alterations encountered in Wistar (Han) rats of this age and strain.
No abnormalities were seen in the reproductive organs of the males and females of Groups 2 (animal nos: 19 and 59) and 4 (animal nos: 36 and 76) that failed to mate, conceive, sire or deliver healthy pups, which could account for their infertility.
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects observed up to the high dose level

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, Parental (Systemic) NOAEL was established at 1000 mg/kg/day.

Executive summary:

A study was conducted to determine the short term repeat dose toxicity of the test substance in rats according to OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Study). Crl:WI(Han) (outbred, SPF-Quality) rats were tested in this study. Males were exposed for 2 weeks prior to mating, during mating, and up to termination. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation. The test substance was administered at 0, 100, 300 and 1000 mg/kg bw/d once daily for 7 d per week. There were no adverse effects on clinical signs, body weight, food consumption and organ weights at necropsy up to the highest tested dose. Macroscopic findings were limited to thickened limiting ridge of the stomach and reddish foci on the stomach glandular mucosa in male rats at 300 and 1000 mg/kg bw/d. Microscopically, hyperplasia and lymphogranulocytic inflammation of the forestomach were observed in the affected males which correlates well with the local irritation effects of repeated dose oral gavage of the test substance. Under the study lconditions, the parental NOAEL for systemic effects were determined to be 1000 mg/kg/day (Zmarowski, 2011).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

A study was conducted to determine the short term repeat dose toxicity of the test substance in rats according to OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Study). Crl:WI(Han) (outbred, SPF-Quality) rats were tested in this study. Males were exposed for 2 weeks prior to mating, during mating, and up to termination. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation. The test substance was administered at 0, 100, 300 and 1000 mg/kg bw/d once daily for 7 d per week. There were no adverse effects on clinical signs, body weight, food consumption and organ weights at necropsy up to the highest tested dose. Macroscopic findings were limited to thickened limiting ridge of the stomach and reddish foci on the stomach glandular mucosa in male rats at 300 and 1000 mg/kg bw/d. Microscopically, hyperplasia and lymphogranulocytic inflammation of the forestomach were observed in the affected males which correlates well with the local irritation effects of repeated dose oral gavage of the test substance. Under the study lconditions, the parental NOAEL for systemic effects were determined to be 1000 mg/kg/day (Zmarowski, 2011).

Study 2:

A study was conducted to determine the short-term repeat dose toxicity of the test substance in rats. Wistar Hoe: WISKf (SPF 71) rats were used in this study. The test substance was administered by oral gavage to male and female rats at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d. The treatment revealed local toxicity in high dose males and females, characterized by macroscopic and microscopic changes in the stomach suggestive of local irritating effects of repeat dose gavage administration. Under the study conditions, no systemic effects were observed in rats up to and including 1000 mg/kg bw/d and the systemic toxicity NOAEL was determined to be 1000 mg/kg bw/d (Anonymous, 1993).

Justification for classification or non-classification

Based on the effects observed in repeated dose toxicity studies, the test substance does not warrant classification for this endpoint according to EU CLP (EC/1272/2008) criteria.