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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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: (P) 13 to 15 weeks old
- Weight at study initiation: (P) Males: 286.4-466.3 g; Females: 200.9-253.4 g
- Fasting period before study: no data
- Housing: in groups of up to five (pre- and post-pairing), one female with one male (paking) or individually (mated females )
- 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 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy

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 and 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-observed adverse-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.

Parental animals: Observations and examinations:

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, on Days 0, 7, 14 and 20 of gestation and on Days 1 and 4post-partum.

OTHER:
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

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

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testes and epididymides of all male adults were weighed at necropsy and qualitative testicular staging with examination of the various cell types present within the different stages of the spermatogenic cycle was performed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, daily clinical observations, individual pup weights on Days 1 and 4 post-partum

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after at least 4 weeks treatment.
- Maternal animals: All surviving animals on Day 5 post-partum and females that did not litter were killed on Day 26 after mating.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Listed tissues from 5 selected adult terminal animals per sex Group 1 and 4adults examined:
Femur+marrow, sciatic nerve, liver, spleen, mesenteric LN, stomach, duodenum, jejunum, ileum, caecum, colon, adrenal, kidney, testis, epididymis, ovary, seminal vesicle, urinary bladder, prostate, uterus, vagina, mandibular LN, thymus, lung, heart, trachea, pituitary, brain, spinal cord, skin+subcutis, pancreas.

Postmortem examinations (offspring):

SACRIFICE
- Surviving pups were killed on Day 5 post-partum
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
GROSS NECROPSY

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 numbers of implantation sites and pups born, the percentage of male pups on Day 1 and the mean pup weights were analysed using non-parametric methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant, the pairwise comparisons were not reported in order to protect the Type I error. The non-parametric methods were used in place of the one-way ANOVA for clinical chemistry parameters with values above or below the l imit 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).

Reproductive indices:

The gestation index and male and female fertility and fecundity indices were analysed using the Cochran-Armitage trend test and Fisher's exact test for pairwise comparisons with control. The tests were interpreted with one-sided risk for a decreased response. The mating index was 100% in all groups and formal analysis was not performed.

Offspring viability indices:

For each litter to day 4 post partum: nuber of pups born (live and dead); daily live litter size and sex, daily clinical observations, individual pup weights on days 1 and 4 post partum

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortality occurred. One high dose group female (number 79) showed signs of subdued bahviour, twitching and piloerection after dosing on Days 4 and 5, was not dosed on Day 6, 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):

High dose males were approximately 9% lighter than controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTIONS
There were no effects of treatment on numbers of males and females mating. One female from each of the control, low and high dose groups was found to be non-pregnant after showing evidence of a positive vaginal smear. There was no treatment-related effect on male or female fertility indices. Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

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.

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:

food consumption and compound intake

other: transient reduction in body weight gain, reduced food intake, reduced forelimb grip strength in males, microscopic findings in the liver of males (centrilobular hypertrophy), kidneys (males: hyaline droplets) and lungs (foamy histiocytes) at 350 mg/kg bw

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

One high dose group female had total embryo foetal loss. One female from the low dose group, one from the intermediate dose group and two from the high dose group had total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly decreeased Day 4 body weights at 350 mg/kg

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

One high dose group female had total embryo foetal loss. One female from the low dose group, one from the intermediate dose group and two from the high dose group had total litter loss. There was an effect of treatment on the high dose pups expressed in a reduced number of implantations, reduced litter size and Day 4 body weights and reduced viability compared to controls. Intermediate dose group pups also had a lower mean body weight on Day 4 compared to controls.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

155 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced numbers of implantations, litter sizes, and Day 4 body weights at 350 mg/kg bw/day

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Conclusions:

Oral administration of the test substance dissolved in 5 % v/v ethanol in corn oil to female and male rats at doses of 70, 155, 350 mg/kg bw/day for 14 days prior to mating, during mating period, gestation and four days post partum had no effects on mating or fertility indices. However, the number of implantations and the litter size were reduced at the highest dose and the NOAEL for reproductive effects is 155 mg/kg bw/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 vehicle (5% v/v ethanol in corn oil) 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. 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 NOAEL for parental toxicity was considered to be 155 mg/kg/day. Oral administration of the test article had no effects on mating or fertility indices. However, the number of implantations and the litter size were reduced at the highest dose and the NOAEL for reproductive effects is 155 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The OECD 422 study is GLP compliant and uses the pure test substance N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, the study follows the requirements of the OECD422 guideline and is therefore of Klimisch 1 validity.

Effect on fertility: via inhalation route

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 Inhalation DNEL for fertility effects following the ECHA guidance with ECETOC modifications. 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. Therefore it is not scientifically justified to test for fertility effects via the inhalation route, this also avoid an unnecessary use of additional animals.

Effect on fertility: via dermal route

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 Dermal DNEL for fertility effects, 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 for fertility effects is not scientifically justified and not performing such a study avoids the unnecessary use of additional animals

Additional information

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, showed no evidence of any adverse effects on fertility when dose to rats up to 350mg/kg bw/day a maximal dose that resulted in some parental toxicity.

 

This information is sufficient without the need for testing by the oral or inhalation routes.

 

Short description of key information:

There is a high quality Klimisch 1 validity GLP OECD422 oral dosing study in rats, this screening study shows no indication of any adverse effects on fertility a relatively high dose levels which showed indication so maternal and paternal toxicity.  There are good scientific arguments that this information is sufficient and that testing by the dermal and inhalation routes would not be scientifically justified.

Justification for selection of Effect on fertility via oral route:

This study is a modern GLP compliant screening study which follows the OECD422 guideline including the well defined test substance, N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4. This study would be expected to detect any clear effects on fertility as both sexes are treated with the test substance for two weeks prior to mating.  This will allow for the calculation of an oral DNEL for any adverse effects on fertility.

Effects on developmental toxicity

Description of key information

There is a high quality Klimisch 1 validity GLP OECD422 oral dosing study in rats, this screening study shows some evidence of developmental toxicity at the highest dose level of 350 mg/kg bw/day in the presence of maternal toxicity, the middle dose of 150 mg/kg bw/day is a NOAEL.  There are good scientific arguments that this information is sufficient and that testing by the dermal and inhalation routes would not be scientifically justified.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-02-16 to 2008-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This is a modern GLP compliant screening study which follows the OECD422 guideline including the well defined test substance, N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4. This study would eb expected to detect any clear developmental toxicity or teratogenic effects as both sexes are treated with the test substance for two weeks prior to mating.

Qualifier:

according to guideline

Guideline:

other: OECD422

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

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

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 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 and until day 4 post-partum, inclusive

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

Details on mating procedure:

After the two week pre-mating dosing the rats were mated one male with each female, daily vaginal smears were taken. The appearance of a positive vaginal smear was taken as an indication of successful mating

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 and until day 4 post-partum, inclusive

Frequency of treatment:

daily

Duration of test:

Males at least 28 days
Females ca. 40 days (until after day 4 post partum.

Remarks:

Doses / Concentrations:
70 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
155 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
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

Maternal examinations:

HISTOPATHOLOGY / ORGAN WEIGHTS
Listed tissues from 5 selected adult terminal animals per sex Group 1 and 4adults examined:
Femur+marrow, sciatic nerve, liver, spleen, mesenteric LN, stomach, duodenum, jejunum, ileum, caecum, colon, adrenal, kidney, ovary, urinary bladder, uterus, vagina, mandibular LN, thymus, lung, heart, trachea,
pituitary, brain, spinal cord, skin+subcutis, pancreas

Ovaries and uterine content:

Ovaries were examined during the post mortem examination and the numbers of implantation sites counted, to allow comparison with the number of offspring seen at day 1 post partum.

Fetal examinations:

Pups were weighed and examined on day 1 post partum for any obvious external abnormalities. They were then observed daily until day 4 when they were again weighed to allow an examination of their weight gain. An assessment was also made of pup survival based on the comparison of the number of pups in each litter as day 1 and Day 4 post partum. Any pups found dead and all pups on day 5 post partum were given a gross post mortem examination for the presence of any abnormalities.

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 numbers
of implantation sites and pups born, the percentage of male pups on Day 1 and the mean pup weights were analysed using nonparametric
methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a
dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant,
the pairwise comparisons were not reported in order to protect the Type I error. The non-parametric methods were used in place of
the one-way ANOVA for clinical chemistry parameters with values above or below the l imit 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).

Indices:

For each litter to day 4 post partum: number of pups born (live and dead); daily live litter size and sex, daily clinical observations,
individual pup weights on days 1 and 4 post partum

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: reduced food intake and effects in the lungs

Details on maternal toxic effects:
No mortality occurred. One high dose group female (number 79) showed signs of subdued bahviour, twitching and piloerection
after dosing on Days 4 and 5, was not dosed on Day 6, staggered after dosing on Day 7 but not on subsequent days.

At histopathological examination, there were several findings, these included in the liver, centrilobular hypertrophy was 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 lungs, there was a minor increase in the level of foamy histiocytes in high dose group 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.

Basis for effect level:

other: Reduced litter size at 350 mg/kg bw/day

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of implantations and the litter size and therefore litter weight were reduced at the highest dose of 350mg/kg bw/day and the NOAEL for developmental effects is 155 mg/kg bw/day. Malformed/shortened limbs occurred in one pup in the intermediate and high dose in the absence in any increase in incidence and severity of this finings with increased dose, these findings are considered to be incidental and not clearly related to treatment

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: Slightly reduced pup survival at 350 mg/kg bw/day

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Oral administration of the test substance dissolved in 5 % v/v ethanol in corn oil to female and male rats at doses of 70, 155, 350 mg/kg bw/day for 14 days prior to mating, during mating period, gestation and four days post partum resulted in limited evidence of developmental toxicity with the number of implantations and the litter size being reduced at the highest dose in the presence of maternal toxic effects, the NOAEL for reproductive effects is 155 mg/kg bw/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 vehicle (5% v/v ethanol in corn oil) 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. 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 findings 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 than adverse, therefore the NOAEL for parental toxicity was considered to be 155 mg/kg/day. Oral administration of the test article had no effects on mating or fertility indices. However, the number of implantations and the litter size were reduced at the highest dose and the NOAEL for reproductive effects is 155 mg/kg bw/day.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The OECD 422 study is GLP compliant and uses the pure test substance N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, the study follows the requirements of the OECD422 guideline and is therefore of Klimisch 1 validity

Effect on developmental toxicity: via inhalation route

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 an inhalation DNEL for developmental toxicity, following the ECHA guidance with ECETOC modifications. 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.
Therefore it is not scientifically justified to test for developmental toxicity via the inhalation route, this also avoid an unnecessary use of additional animals.

Effect on developmental toxicity: via dermal route

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 Dermal DNEL for developmental toxicity 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 dermal testing for developmental toxicity is not scientifically justified and not performing such a study avoids the unnecessary use of additional animals

Additional information

The OECD422 study showed some indication of developmental toxicity in the form of reduced litter size and reduced viability at the highest dose level of 350 mg/kg bw/day. Malformed/shortened limbs occurred in one pup in the intermediate and high dose in the absence in any increase in incidence and severity of this finings with increased dose, these findings are considered to be incidental and not clearly related to treatment.

 

There were no indications of any teratogenic effects seen in the offspring when given gross postmortem examinations on day 5 post partum. More subtle effects that might have occurred such as effect on skeletal ossification cannot be assessed in this screening study. The NOAEL of 150mg/kg bw/day is considered suitable for the calculation of an oral DNEL for developmental toxicity. DNELs calculated from this oral NOAEL would be expected to be protective for dermal exposure due to the very low dermal penetration and for inhalation in part due to the low potential for exposure by this route.

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

This study is a modern GLP compliant screening study which follows the OECD422 guideline including the well defined test substance, N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4. This study would be expected to detect any clear developmental toxicity or teratogenic effects as both sexes are treated with the test substance for two weeks prior to mating.  This will allow for the calculation of an oral DNEL for any developmental toxicity seen.

Justification for classification or non-classification

There were no findings in the OECD422 study on N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, to indicate any adverse effects on fertility, therefore there is no requirement for a classification for toxicity to reproduction for such effects. There were some indications of developmental toxicity seen at the highest dose of 350 mg/kg bw/day, but these were indicative of embryotoxicity and also could have resulted to some extent from poor maternal neutering of the offspring due to the maternal toxic effects seen at that dose level. There were no indications of any structural abnormalities seen within the limitations of this screening study or any post-natal functional deficiencies. Based on this and the screening nature of this study the effects seen only in the highest dose are not considered sufficient to justify classification for toxicity to reproduction/developmental toxicity.