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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the Ames test and the Mouse lymphoma in-vitro tests and the in-vivo mouse micronucleus test.  All three tests were negative.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1998-04-11 to 1998-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study performed under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl:CD-1 (ICR) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: approximately 8 weeks (dose range finding 1 and micronucleus assay), approximately 9 weeks (dose range finding 2),
- Weight at study initiation: 28.8 - 35.8 g (males, dose range finding 1), 21.6 - 25.0 g (females, dose range finding 1), 34.1-35.5 g (males, dose range finding 2), 22.8 - 27.8 g (females, dose range finding 2), 31.4 - 38.4 g (micronucleus assay)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of up to 5 animals in sanitary polycarbonate cages containing bedding
- Diet (e.g. ad libitum): PMI Feeds, Inc., Certified Rodent Diet # 5002 ad libitum
- Water (e.g. ad libitum): tap water ad liblitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 26.1
- Humidity (%): 30-70
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 1998-03-24 to 1998-04-11

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 20, 50, 80, 150, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS (micronucleus assay): Prior to dosing, the top stock solution of the test article was prepared by adding the appropriate volume of the vehicle, corn oil , to a preweighed quantity of the test article. The stock was mixed by stirring with a spatula for about 7 minutes, homogenising for about 3 minutes, and stirring on a plate for about 2 minutes. The remaining stocks were prepared by dilution from the top stock. The stocks were cloudy, slightly viscous, light yellow oil suspensions with small evenly distributed particles. All dosing stocks were continuously mixed on a plate during the dosing procedure and protected from light during preparation and dosing.

Duration of treatment / exposure:

single oral dose

Frequency of treatment:

single oral dose

Post exposure period:

175 mg/kg: 24 hour treatment
350 mg/kg: 24 hour treatment
700 mg/kg: 24 and 48 hour treatment
Vehicle control: 24 and 48 hour treatment
Positive control: 24 hour treatment

Remarks:

Doses / Concentrations:
dose range finding 1: 200, 500, 800, 1500, 2000 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
dose range finding 2: 600, 700 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
micronucleus assay, 24 h preparation interval: 173, 350, 700 mg/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
micronucleus assay, 48 h preparation interval: 700 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

dose range finding 1: 3 males and 3 females per dose
dose range finding 2: 3 males and 3 females per dose (One female of the 2000 mg/kg group not dosed due to breakage of the dosing stock bottle)
micronucleus assay: 6 males per dose and harves timepoint

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): not reported, but substance proposed in OECD TG 474
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Tissues and cell types examined:

- clinical signs: examination for toxic signs and mortalities at least daily
- bone marrow cells: sampling at 24 or 48 hours after treatment

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results of both dose range finding studies (some mortality at the 700 mg/kg dose level and high mortality at the 800 mg/kg dose level), the maximum tolerated dose was estimated to be 700 mg/kg. Based on these results, dose levels of 175, 350 and 700 mg/kg were selected for testing in this study. Since there were no differences in toxicity observed between the sexes, only male mice were used for the micronucleus study.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest timepoints, the animals were euthanized by CO2 inhalation followed by incision of the diaphragm. The hind limb bones (tibias or femurs) were removed from the first 5 surviving animals for marrow extraction. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3-5 mL fetal bovine serum (one tube per animal). Animals in excess of the first 5 survivors were euthanized but no marrow was extracted. Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grünwald Solution followed by Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

METHOD OF ANALYSIS:
The micronucleus frequency (expressed as percent micronucIeated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The frequency of PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 200 erythrocytes on the slide. Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1120 and 115 the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than 1 micronucleus was counted as 1 micronucleated PCE, not 2 (or more) micronuclei .

Evaluation criteria:

The criteria for a positive response was the detection of a statistically significant positive response for at least 1 dose level and a statistically significant dose related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response, the study director also considered the biological relevance of the results in the final evaluation.

Statistics:

Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p < 0.05), a Dunnett's t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time. Additionally, parametric or nonparametric tests for trend may have been employed to identify any dose-related response.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

signs of clinical toxicity in animals treated with 350 and 700 mg/kg and cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at 700 mg/kg, at the 48 hour harvest timepoint

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 - 2000 mg/kg
- Solubility: small evenly distributed particles observed in suspension
- Clinical signs of toxicity in test animals: See table in "Any other information on results incl. tables"
- Evidence of cytotoxicity in tissue analyzed: No tissue analyzed
- Rationale for exposure: not reported
- Gender specific differences in toxicity: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): N-tert-Octylacrylamide induced no statistically significant increases in micronucleated polychrornatic erythrocytes over the levels observed in the vehicle controls at any of the harvest timepoints.
- Ratio of PCE/NCE (for Micronucleus assay): cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at 700 mg/kg, at the 48 hour harvest timepoint
- Appropriateness of dose levels and route: signs of clinical toxicity observed, cytotociy to the bone marrow at 700 mg/kg at 48 harvest timepoint

Table 1: Toxicity observed in dose range finding test

Target Dose Level (mg/kg)	Sex	Time after dosing
		IPD	1 h PD	1 day	2 days
200	M	1, 2	0	0	14
		1, 2	5	2, 13, 14	2, 13, 14
		1, 2	5	2, 13, 14	2, 13, 14
	F	1, 2, 3	1	0	0
		1, 2	1	0	0
		1, 2	1	0	0
500	M	1, 2, 5	2, 3, 4, 5, 7	2, 13, 14	2, 13, 14
		1, 2, 5	3, 4, 5, 7	2, 13, 14	2, 13, 14
		1, 2, 5	2, 4, 5	2, 13, 14	2, 13, 14
	F	2, 4	3, 7, 9	0	0
		4, 5, 6	7, 9	4, 5, 8	0
		1, 2	2, 4, 5, 7	2, 3, 4	0
600	M	4, 15	3, 9	2, 14	0
		4	3, 4, 15	2, 14	0
		4	3, 4, 15	2, 14	0
	F	15	3, 9, 16	3, 4, 6, 14, 15	0
		15	3, 9, 16	14	0
		15	3, 4, 7, 15	14	0
700	M	15	3, 4, 15	14, 17	0
		15	3, 9	14	0
		15	3, 6	2, 13, 14	0
	F	15	3, 4, 15	14	0
		15	3, 9	4, 7, 14, 15	0
		0	3, 7, 9	11	-
800	M	4, 5, 7, 8	3, 7, 8, 9	0	-
		4, 5, 7, 8	3, 7, 8, 9	4, 5, 8	-
		1, 2, 5, 7	2, 4, 5, 7, 8	2, 3, 4	14
	F	4, 5, 6	3, 7, 8, 9	11	13, 14
		3, 4, 5	7, 8, 9	11	-
		2, 5, 8	7, 9	2, 3, 4, 5, 13, 14	13, 14
1500	M	4, 5, 6, 7, 8	7, 9, 10	11	-
		4, 5, 7, 8	7, 9, 10	11	-
		4, 5	7, 9, 10	11	-
	F	3, 6, 7, 9, 10	7, 9, 10	3, 6, 7, 9, 10	4, 5, 6, 8
		3, 6, 7, 9, 10	6, 7, 9, 12	11	-
		3, 6, 7, 9, 10	7, 9, 10	11	-
2000	M	1, 5	1	1, 2, 13, 14	13, 14
		4, 5, 8	8, 9	2, 4, 13, 14	2, 13, 14
		7, 9, 10	11	-	-
	F	11	-	-	-
		7, 9, 10	11	-	-

Key: 0=Normal, 1=slightly hypoactive, 2=hunchedposture, 3=squintedeyes, 4=hypoactivem, 5=ataxic, 6=cold to touch, 7= tremors, 8=polypnea, 9=prostrate, 10=dyspnea, 11=found dead, 12=gasping, 13=rough haircoat, 14=distended abdomen, 15=ataxic, 16=convulsions, 17=fecal stains

IPD=immediately post dosing last animal,PD=post dosing last animal

Table 2: Toxicity observed in micronucleus assay

Target Dose Level (mg/kg)	Harvest time point	Time after dosing
		IPD	1 h PD	1 day	2 days
350	24	1	0	0	-
		1	0	0	-
		1	0	0	-
		1	0	0	-
		1	5, 7	0	-
		1	0	0	-
500 600	24	2	2, 3, 7	0	-
		2	2, 3, 7	0	-
		2	2	0	-
		2	2, 3, 6, 7	0	-
		2	2, 3, 7	0	-
		2	2, 3, 7	0	-
	48	2	2, 6, 7, 8	0	0
		2	2, 3	0	0
		3	2, 3	0	0
		3	6, 9	0	0
		2	2, 3	0	0
		2	2, 3	0	0
	Secondary	2	1	0	0
		3	9, 10	0	0
		2	2, 3	0	0
		3	2, 3	0	0
		2	2, 3, 10	0	0
		2	3, 7	0	0

Key:0=, 1=slightly hypoactive, 2=hypoactive, 3=ataxic,5=urinestains,6=squintedeyes,7=hunched posture, 8=polypnea, 9=prostrate, 10=tremors

IPD=immediately post dosing last animal, PD=post dosing last animal

Micronucleus data

Treatment	Dose	Harvest time	% micronucleated PCEs mean of 2000 per animal ± SE Males	Ratio PCE:NCE Mean ± SE Males
Vehicle control	Corn oil	24 h	0.05 ± 0.03	0.42 ± 0.02
Vehicle control	Corn oil	48 h	0.02 ± 0.02	0.48 ± 0.06
Positive control	Cyclophpsphamide 80 mg/kg	24 h	3.78 ± 0.15 *	0.41 ±0.05
Test article	175 mg/kg	24 h	0.00 ± 0.00	0.30 ± 0.02
Test article	350 mg/kg	24 h	0.03 ± 0.02	0.34 ± 0.04
Test article	700 mg/kg	24 h	0.05 ± 0.02	0.36 ± 0.06
Test article	700 mg/kg	48 h	0.06 ± 0.01	0.31 ± 0.04 **

* Significantly greater than the corresponding vehicle control, p<0.01

** Significantly less than the corresponding vehicle control, p<= 0.05

Conclusions:

Interpretation of results (migrated information): negative
The test article, N-tert-Octylacrylamide, induced signs of clinical toxicity in the treated animals and was cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at 700 mg/kg, at the 48 hour harvest timepoint. N-tert-Octylacrylamide did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes and is considered negative in the mouse bone marrow micronucleus test under the conditions of exposure in this assay.

Executive summary:

The objective of this study was to evaluate the test article, N-tert-Octylacrylamide, for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte cells in Crl:CD-1 (ICR) BR mouse bone marrow. The study was conducted according to the principles of GLP and according to the methods outlined in OECD TG 474.

In the initial dose range finding study, the test article was suspended in corn oil and dosed by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 200, 500, 800, 1500 and 2000 mg/kg (only 2 females were dosed at 2000 mg/kg) and observed for 2 days after dosing for toxic signs and/or mortality. In the second dose range finding study, the test article was suspended in corn oil and dosed by oral gavage to 3 males and 3 females per dose level. The animals were dosed at 600 and

700 mg/kg and observed for 2 days after dosing for toxic signs and/or mortality. Based on the results of the dose range finding studies, the maximum tolerated dose was estimated to be 700 mg/kg. Since there were no differences in toxicity observed between the sexes, only male mice were used for the micronucleus study. In the micronucleus assay, the test article was suspended in corn oil and dosed by oral gavage to 6 males per dose level and harvest timepoint. The animals were dosed at 175, 350 and 700 mg/kg. The first 5 surviving animals dosed at the 175 and 350 mg/kg dose levels and with the positive control were euthanised approximately 24 hours after dosing for extraction of the bone marrow . The first 5 surviving animals dosed at the 700 mg/kg dose level and with the vehicle control were euthanised approximately 24 and 48 hours after dosing for extraction of the bone marrow.

The test article, N-tert-Octylacrylamide, induced signs of clinical toxicity in the treated animals and was cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at 700 mg/kg, at the 48 hour harvest timepoint. N-tert-Octylacrylarnide did not induce a statistically significant increase in micronuclei in bone marrow polychrornatic erythrocytes and is considered negative in the mouse bone marrow micronucleus test under the conditions of exposure in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the Ames test and the Mouse lymphoma in-vitro tests and it was found not to be mutagenic. An in-vitro cytogenetics test was not performed as there is an in-vivo mouse micronucleus study available. This study showed no indications of clastogenicity despite there being clear signs of cytotoxicity in the bone marrow, indicating that the test substance or its metabolites were reaching the bone marrow.

Justification for selection of genetic toxicity endpoint

This study is an in-vivo test and as cytotoxicity was seen in the bone marrow test substance or its metabolites had reached the bone marrow.  The study confirmed in-vivo the lack of genotoxicty in the two in-vitro tests.

Justification for classification or non-classification

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the Ames test and the Mouse lymphoma in-vitro tests and the in-vivo mouse micronucleus test. All three tests were valid and negative, indicating no potential for mutagenicity or clastogenicity. Therefore the test substance is not classified for mutagenicity.