N-(1,1,3,3-tetramethylbutyl)acrylamide

Endpoint summary

Description of key information

The is an OECD422 Oral gavage dosing study available which is GLP compliant and Klimisch 1 rated for validity.  This study is suitable as the basis for deriving DNELS based on the NOAEL values determined.  The low vapour pressure and low dermal penetration support the use of the oral NOAEL for the calculation of DNELs for oral, dermal and inhalation exposure

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2004-02-16 to 2008-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(GIx/BRL/Han)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate
- Age at study initiation: 13 to 15 weeks old
- Weight at study initiation: from 286.4 to 466.3 g (males) and from 200.9 to 253.4 g (females)
- Fasting period before study: no data
- Housing: in groups of up to five (pre- and post-pairing)
- Diet (e.g. ad libitum): SQC Rat and Mouse Breeder Diet No 3, Expanded. (Special Diets Sevices Ltd. Witham), ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: at least 10 days prior to the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 5 % v/v ethanol in corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 14, 31 and 70 mg/mL of t-octyl acrylamide
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC: analysis was performed at the beginning and at the end of the dosing period

Duration of treatment / exposure:

Males: for 2 weeks prior to pairing, during the pairing period and until the day before necropsy
Females: for 2 weeks prior to pairing, during the pairing period ans until day 4 post-partum, inclusive

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 70, 155, 350 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the high dose was based on the results of the rat Maximum Tolerated Dose assay where a dose level of 300 mg/kg/day was the no-observedadverse-effect-level. Dose levels of 400 mg/kg/day or above caused neurological signs such as ataxia, prostration, spasmodic twitching and convulsions in some females and males. The low dose level of 70 mg/kg/day was not expected to elicit parental toxicity and the intermediate dose level of 155 mg/kg/day is the geometric mean of these values.
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale (if not random): no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (am/pm) for morbidity/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (am/pm)

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly for the males. For the females, individual body weights were recorded weekly prior to pairing and until confirmation of mating.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 5 males and 5 females per group
- Following parameters were examined: haemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocytes, total and differential white cell count, platelet count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: 5 males and 5 females per group
- Following parameters were examined: aspartate aminotransferase, alkaline phosphatase, potassium, inorganic phosphorus, total protein, globulin, total cholesterol, urea, creatinine, alanine aminotransferase, sodium, calcium, chloride, albumin, albumin/globulin ratio, glucose, total bilirubin

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of the treatment period of the males and during the lactation period of the females
- Dose groups that were examined: from each group
- Battery of functions tested: grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body weight gains, necropsy body weights, food consumption, haematology, clinical chemistry, locomotor and functional observation data were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed Where this showed no evidence of heterogeneity (P≥0.01). pairwise comparisons with control were made using Dunnett's test. A linear contrast was used to determine whether there was a relationship between increasing dose and response. A significant trend (P<0.05) was reported only where none of the pairwise comparisons was significant. The non-parametric methods were used in place of the one-way ANOVA for clinical chemistry parameters with values above or below the limit of the assay. Organ weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett's test, for each sex separately, using the necropsy body weight as covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where the test for equality of slopes failed (male heart weights; P<0.01). The organ was analysed using one-way ANOVA on absolute organ weights and organ to necropsy body weight ratios. Levene's test for equality of variances across the groups was also performed for all organs and, in all cases, this showed no evidence of heterogeneity (P≥0.01).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortality occured. One high dose group female (number 79) showed signs of subdued behaviour, twitching and piloerection after dosing on Days 4 and 5. This animal was not dosed on Day 6. This animal staggered after dosing on Day 7 but not on subsequent days.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality occured. One high dose group female (number 79) showed signs of subdued behaviour, twitching and piloerection after dosing on Days 4 and 5. This animal was not dosed on Day 6. This animal staggered after dosing on Day 7 but not on subsequent days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose males were approximately 9 % lighter than controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the pre-pairing period, high dose males and females had reduced mean food intakes compared to controls. Mean food intakes for the high dose females were also slightly lower than controls during gestation.

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:

effects observed, treatment-related

Description (incidence and severity):

These findings are of uncertain significance or not considered to be ralated to treatment.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Forelimb grip strength in male rats during Week 7 showed a statistically significant (P<0.05) dose-related decrease. This was considered to be a possible effect of treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Gross pathological findings:

no effects observed

Description (incidence and severity):

no adverse effects of treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see section "Details on results"

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occured. One high dose group female (number 79) showed signs of subdued behaviour, twitching and piloerection after dosing on Days 4 and 5. This animal was not dosed on Day 6. This animal staggered after dosing on Day 7 but not on subsequent days.

BODY WEIGHT AND WEIGHT GAIN
The majority of males in the high dose group lost body weight during the fmt week of dosing. Although mean body weights increased every week after that, the high dose males were still approximately 9 % lighter than controls by the end of the study.

OPHTHALMOSCOPIC EXAMINATION
no data

HAEMATOLOGY
There was a slight increase (P<0.05) in absolute numbers of reticulocytes and platelets in the high dose group males, but this increase was not seen in females. The toxicological significance of this finding is uncertain. There was a slightly increased number of neutrophils in the low dose group males (P<0.05), but as there was no increase in the intermediate or high dose group males, this slight increase was not deemed to be related to treatment.

CLINICAL CHEMISTRY
There was a statistically significant increase in total protein in the intermediate and high dose group males (P<0.01), and high dose group females (P<0.001). Aspartate aminotransferase levels were increased in high dose group females (P<0.05). These findings are of uncertain significance. Other parameters occasionally achieved statistical significance due to the large intraanimal variation seen in rats and were not considered to be related to treatment.

URINALYSIS
no data

NEUROBEHAVIOUR
Forelimb grip strength in male rats during Week 7 showed a statistically significant (P<0.05) dose-related decrease. This was considered to be a possible effect of treatment.

ORGAN WEIGHTS
Adjusted mean liver weight was inaeased in all the treated male groups (P0<0.05, P<0.001, P<0.001 for the low, intermediate and high dose groups
respectively) and in the intermediate and high dose group females (P<0.01, P<0.001 respectively). Adjusted mean adrenal weight was increased in high dose group females (P<0.01). These were considered to be effects of treatment.

GROSS PATHOLOGY
No adverse effects of treatment. There was a minor increase in pale and mottled kidneys in the males from all the treated groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver, centrilobular hypertrophy was recorded for the majority of intermediate dose group males and all high dose group males. The same finding was recorded for two intermediate dose group females and the majority of high dose group females. Centrilobular hypertrophy was characterised by enlarged hepatocytes with a pale or more eosinophilic cytoplasm.
In the kidney, there was an increase in the level of hyaline droplets in all high dose group males. This was characterised by the presence of densely eosinophilic, variably-sized droplets in the cytoplasm of proximal tubular cells. An associated increase in the level of focal nephropathy was also recorded in the high dose group males, characterised by the presence of basophilic tubules in the inner cortex/outer medulla. This male ratspecific hyaline droplet nephropathy is of little relevance to risk assessment in humans.
In the lungs, there was a minor increase in the level of foamy histiocytes in high dose group males and females. Foamy histiocytes were characterised by small collections of alveolar histiocytes with abundant foamy cytoplasm.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: at 350 mg/kg/day: reduced food intakes, a transient reduction in body weight gain, reduced forelimb grip strength in males and microscopic fidings in the liver, kidneys and lungs

Critical effects observed:

not specified

Conclusions:

Oral administration of t-octyl acrylamide at a dose level of 350 mg/kg/day elicited adult toxicity in the fom of reduced food intakes, a transient reduction in body weight gain, reduced forelimb grip strength in males and microscopic fidings in the liver, kidneys and lungs. At a dose level of 155 mg/kg/day liver weights in male rats was increased with microscopic liver changes seen in some males and females. This change is considered adaptive rather then adverse, therefore the no-observable-adverse-effect level (NOAEL) for t-octyl acrylamide was considered to be 155 mg/kg/day.

Executive summary:

The purpose of this study was to provide preliminary information on the possible effects of the test article t-octyl acrylamide following repeated administration in the rat following OECD Guideline 422. Male and female Crl:WI(Glx/BRL/Han)BR rats were assigned to four groups (10 animals/sex/group). Each treated group received dose preparations containing the control article or 70, 155 or 350 mg of test article/kg of body weight/day (mg/kg/day) at a dose volume of 5 mL/kg. Animals were treated for two weeks prior to mating, during mating period of 15 days and in case of females during gestation and for 4 days post partum.On Day 4 of dosing, one high dose group female had post-dosing neuro-behavioural signs at 2 and 4 hours after dosing. On Day 5 of dosing, the same female had postdosing neuro-behavioural signs up to 4 hours after dosing. This animal was not dosed on Day 6 of treatment and on Day 7 was observed as staggering immediately after dosing. The majority of males in the high dose group lost body weight during the first week of dosing. During the pre-pairing period, high dose males and females had reduced mean food intakes compared to controls. Forelimb grip strength in male rats during Week 7 showed a slight dose-related decrease. This was considered to be a possible effect of treatment but the lack of corroborating fmdings in the functional observation battery suggest that this observation was not due to neurotoxicity caused by the test article. There was a slight increase in absolute numbers of reticulocytes and platelets in the high dose group males, but this increase was not seen in females. The toxicological significance of this finding is uncertain. There was an increase in total protein in the intermediate and high dose group males, and high dose group females. Aspartate aminotransferase levels were increased in high dose group females. These findigs are of uncertain significance. Adjusted mean kidney weight was increased in high dose group males and also increased slightly in females in a dose-related manner. Adjusted mean liver weight was increased in all the treated male groups and in the intermediate and high dose group females. Adjusted mean adrenal weight was increased in high dose group females. These were considered to be effects of treatment. Macroscopically, there was a minor increase in pale and mottled kidneys in the males from all the treated groups. In the liver, centrilobular hypertrophy was recorded for all high dose group males and females. In the kidney, there was an increase in the level of hyaliie droplets, with an associated increase in focal nephropathy, in all high dose group males. In the lungs, there was a minor increase in the level of foamy histiocytes in high dose group males and females. Oral administration of t-octyl acrylamide at a dose level of 350 mg/kg/day elicited adult toxicity in the form of reduced food intakes, a transient reduction in body weight gain, reduced forelimb grip strength in males and microscopic fidiigs in the liver, kidneys and lungs. At a dose level of 155 mg/kg/day liver weights in male rats was increased with microscopic liver changes seen in some males and females. This change is considered adaptive rather then adverse, therefore the no-observable-adverse-effect level (NOAEL) for t-octyl acrylamide was considered to be 155 mg/kg/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

155 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available OECD422 study on N-(1,1,3,3-tetramethylbutyl)acrylamide is a high quality study to GLP with a Klimisch rating of 1.

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

Quality of whole database:

The lack of irritancy to skin and mucous membranes based on in-vitro testing, supported by low irritant potential in the mouse local lymph node assay indicate that local irritant effects in the respiratory tract are not expected. Therefore the NOAEL from the oral OECD422 study in rats is suitable for the calculation of long-term inhalation DNELs that can be expected to also protect from local effects in the respiratory tract.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

There is a high quality Klimisch 1 validity GLP OECD422 oral dosing study in rats, which allows for the calculation of a long term Dermal systemic DNEL following the ECHA guidance with ECETOC modifications. The dermal penetration study shows very low dermal penetration certainly less than 10% and possibly as low as 0.3% in rats. Based on this the calculated DNEL based on the oral study will be very conservative and will certainly protect from any possible toxic effects from dermal exposure. Therefore repeat dose dermal toxicity testing is not scientifically justified and not performing such a study avoids the unnecessary use of additional animals

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

The lack of irritancy to skin and mucous membranes based on in-vitro testing, supported by low irritant potential in the mouse local lymph node assay indicate that local irritant effects in the respiratory tract are not expected. Therefore the NOAEL from the oral OECD422 study in rats is suitable for the calculation of long-term dermal DNELs that can be expected to also protect from local dermal effects.

Additional information

The effects seen in the OECD422 study were primarily in the 350mg/kg males of effects in the kidneys of hyaline droplets in the cytoplasm of the proximal tubular cells with an associated increase in the level of local nephropathy. This was characterized by the presence of basophilic tubules inner cortex/outer medulla. This male rat specific hyaline droplet nephropathy is considered to be of little relevance to humans. There were some minor effect in the lungs in the 350 mg/kg/day males and females characterized as small collections alveolar histiocytes with abundant foamy cytoplasm. The other main finding was in the liver of the majority of the 155 mg/kg/day and 350 mg/kg/day males, two 155mg/kg/day and the majority of the 350mg/kg/day females, which is considered not to be adverse but to be due to enzyme induction for the metabolism of the xenobiotic test substance. Males also showed a dose depended decrease in forelimb grip strength, which was not accompanied by any other dose related observations on functional parameters. There were no effects on locomotor activity at any dose level which indicated that the effects seen on forelimb grip strength was not due to nerotoxicty of the test substance. As the affects seen in the liver at 155mg/kg/day were not considered to be adverse a NOAEL was determined to be 155mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The is an OECD422 Oral gavage dosing study available which is GLP compliant and Klimisch 1 rated for validity.  This study is suitable as the basis for deriving DNELS based on the NOAEL values determined

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

There is a high quality Klimisch 1 validity GLP OECD422 oral dosing study in rats, which allows for the calculation of a long term systemic inhalation DNEL following the ECHA guidance with ECETOC modifications. N-(1,1,3,3-tetramethylbutyl)acrylamide is not irritant to the skin and mucous membranes, so it can be assumed that there would be no significant local irritant effects in the respiratory tract.  Based on this the long term systemic DNEL is considered to also protect from any local effect in the respiratory tract.  In addition the relatively low vapour pressure of N-(1,1,3,3-tetramethylbutyl)acrylamide will limit the possibility of inhalation exposure to vapour and in Europe this monomer is only handled incorporated in polymers with residual monomer less than 0.1% so there is very little possibility of inhalation exposure.  No study is scientifically justified.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

The NOAEL in the OECD 422 study of 155mg/kg/day was for gavage dosing in males for 28 days and females for up to ca.40 days. Systemic toxic effects were only seen in the 350mg/kg/day group. The EU CLP (GHS) criteria for classification for Specific Target Organ Toxicity (STOT) are based on data from a 90 day study, for Category 2 the range for such effects is 10-<100 mg/kg/day. Where the data are from a 28 day study these levels are multiplied by three. In this OECD study as the dosing was for 28 – ca. 40 days depending on the sex of the rats, these limits are multiplied by three to 30-<300 mg/kg/day. As the highest dose in this study was 350mg/kg/day it could potentially require a classification for category 2 for STOT if effects were seen in the 155mg/kg/day group.

Based on these findings there is no evidence of relevant specific target organ toxicity in the rats dosed at up to the middle dose level of 155mg/kg/day, an even at 350mg/kg/day the effects seen in the male kidneys are not relevant to humans, therefore there is no requirement for a classification for STOT under the EU CLP (GHS) criteria