Docosyl methacrylate

Endpoint summary

Description of key information

No studies on repeated oral dose toxicity for the target substance Docosyl methacrylate are available. Therefore, read-across from studies with the primary metabolite Docosan-1-ol (Behenyl alcohol, CAS 661-19-8) as well as with the structurally analogous substance Dodecyl methacrylate (CAS 142-90-5) was performed.

Oral: Daily oral administration of Dodecyl methacrylate to Sprague-Dawley rats in a study according OECD TG 422 did not elicit any signs of systemic toxicity up to the highest dose tested. The NOAEL for parental toxicity was considered to be 1000 mg/kg bw/day. In two subchronic studies with Behenyl alcohol (Docosan-1-ol), no adverse effects were observed up to the highest dose tested. The NOAEL for male and female rats was 1000 mg/kg bw/day and for dogs of both sexes the NOAEL was 2000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

Beagle dogs were administered Docosan-1-ol (Behenyl alcohol) by oral gavage for 27 weeks at doses of 0, 20, 200, or 2000 mg/kg bw/day.

GLP compliance:

yes

Remarks:

Huntingdon Life Sciences Ltd (Suffolk, England)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Consort Limited Harewood Park (Hereford, England)
- Age at study initiation: 15 to 19 weeks
- Housing: individually housed in indoor kennels equipped with under floor heating, solid partitions, sawdust bedding, food hoppers, and water
dispensers that were changed at appropriate intervals
- Diet: expanded dog diet (Special Diets Services, Witham, Essex, England),ad libitum
- Water: tap water, ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

other: 1% w/w aqueous Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed into a glass container and heated (approximately 80°C) until molten using an electric mantle.
An appropriate volume of vehicle (1% Tween 80) was heated in a water bath to at least 75°C and then combined with the molten behenyl alcohol under continuous magnetic stirring, to a concentration of 20% behenyl alcohol. The resulting suspension was slowly cooled, with homogenization to a temperature of below 60°C, and then further cooled in a water bath to a temperature of 30°C. The 20% suspension was prepared once each week. Lower concentrations were prepared on the day of use by dilution of the 20% suspension with 1% w/w aqueous Tween 80.

VEHICLE
- aqueous Tween 80
- Amount of vehicle (if gavage): 1%

Animals received the test material or vehicle control formulations by oral gavage, at a volume-dosage of 10 mL/kg body weight, using a 30 choke plastic catheter.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

27 weeks

Frequency of treatment:

7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 male and 4 female per dose

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

Dogs were subjected to a rigorous veterinary examination prestudy, and after 11 and 24 weeks of the study
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prestudy, weekly for the duration of the study, and at necropsy

FOOD CONSUMPTION
- calculatedweekly throughout the treatment periods

FOOD EFFICIENCY:
- Weekly group mean food conversion efficiencies were calculated for the first 14 weeks of the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prestudy, and during weeks 12 and 25 of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prestudy and after weeks 12 and 25 of the study
- Anaesthetic used for blood collection: not specified
- Animals fasted: Yes
- How many animals: all
- Parameters checked: hemoglobin concentrations, erythrocyte count, total and differential leukocyte count, platelet count, mean cell hemoglobin concentration, mean cell hemoglobin, morphology and unusual cell types, including normoblasts, anticoagulant and prothrombin time was measured asn activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Parameters checked: alkaline phosphatase activity, alanine amino-transferase, aspartate amino-transferase,gamma-glutamyl transpeptidase, glucose, total bilirubin, total cholesterol, urea, total triglyceride, total protein,and electrolyte levels: Na, K, Cl, and C, inorganic phosphorus and electrophorectic protein, creatine phosphokinase activity

URINALYSIS: Yes
- Time schedule for collection of urine: during weeks 12 and 25
- Metabolism cages used for collection of urine: No
- Animals fasted: yes
- Parameters checked: urine pH and protein, glucose, ketones, bilirubin, urobilinogen, blood, urine-specific gravity, the appearance and volume of urine, nitrites and total reducing substances

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- detailed examination of the external features and orifices, the neck and associated tissues, and the cranial, thoracic, abdominal, and pelvic cavities, and their viscera

HISTOPATHOLOGY: Yes
- adrenals, brain, eyes and optic nerve, heart, kidneys, liver, lungs, spinal cord, stomach, thyroid, and uterus

Statistics:

For organ weights and body weight changes, homogeneity of variance was evaluated by Bartlett’s test. Whenever this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons; otherwise, a Dunnett’s test was used. Fisher’s exact test was applied as a two-tailed test, where appropriate, to the distribution of macroscopic or microscopic pathological entities. Unless stated, group mean values or incidences for the treated groups were not significantly different from those for the controls (P>0.05). The significance of intergroup differences in hematology (excluding the incidence of morphological abnormalities evident on the blood smears for rats), blood chemistry, and urinalysis (volume, pH, and specific gravity only) was assessed by Student’s t test using a pooled error variance.
As a small number of dogs were used in the study, the statistical results cannot be considered definitiveand are only used as a guide in the interpretation ofthe results.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pale feces in all dogs treated with 2000 mg/kg bw/day were detected. One male and 3 females treated with 200 mg/kg bw/day
were observed to have pale feces on several occasions. One control male had pale feces on a single occasion. Pale feces are likely associated with the presence of unabsorbed test item in the GI being passed in the stools of these animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Toxikokinetic study:
Blood sample results were obtained from dogs on Day 1, and during Weeks 13 and 26 of the study. No statistically significant sex-related differences in systemic exposure occurred in any of the dose groups (P=0.576). The times at which the maximum plasma concentrations occurred, i.e., Cmax, ranged from 2 to 16 h post-dose, and were independent of dose level, sex, and day of sampling. At the highest dose tested, the Cmax and AUC24 values ranged from 1094 to 2860 ng/mL and 11,830 to 27,830 ng x h/mL, respectively, throughout the duration of the study. The rate and extent of systemic exposure of dogs to behenyl alcohol, characterized by the Cmax and AUC24, respectively, increased with increasing dose level; however, these increases were less than the proportionate dose increment. There was statistically significant evidence of non-proportionality for Cmax on all sampling days (P=0.005), and for AUC24during Weeks 13 and 26 (P<0.001).

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Critical effects observed:

not specified

Table 1: Maximum Mean Plasma concentrations of Behenyl Alcohol in dogs and the areas under the mean plasma behenyl alcohol concentration time curves estimated up to 24 h postdose on Day 1 and during weeks 13 and 26 of the study

	Day 1		Week 13		Week 26
Dose level (mg/kg bw/day)	Males	Females	Males	Females	Males	Females
	Maximum mean plasma concentration (Cmax) (ng/mL)
20	74 (50)	26 (10)	159 (91)	151(44)	137 (53)	310 (205)
200	473 (244)	207 (93)	1841 (601)	1186 (854)	757 (388)	1289 (1020)
2000	1308 (326)	1469 (463)	2140 (272)	2860 (1077)	1094 (814)	2255 (718)
	Area under the curve (AUC24)(ng x h/mL)
20	610 (473)	177 (115)	1550 (835)	1377 (449)	848 (434)	2031 (1073)
200	4419 (1856)	1781 (1166)	14370 (3755)	8425 (6533)	7435 (3575)	7799 (5492)
2000	11830 (3238)	15080 (7426)	22430 (2275)	27830 (8361)	12340 (9028)	24400 (8043)

Numbers in parentheses represent standard deviations and standard errors of the Cmax and AUC24, respectively.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

CD rats were administered Docosan-1-ol (Behenyl alcohol) by oral gavage for 26 weeks at doses of 0, 10, 100, or 1000 mg/kg bw/day.

GLP compliance:

yes

Remarks:

Huntingdon Life Sciences Ltd (Suffolk, England)

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited (Margate, Kent, England)
- Age at study initiation: 21 to 28 days
- Housing: stainless-steel cages, male and female rats were separated and housed 5 per cage
- Diet: expanded rodent diet without added antibiotic or other chemotherapeutic or prophylactic agent, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

other: 1% w/w aqueous Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed into a glass container and heated (approximately 80°C) until molten using an electric mantle.
An appropriate volume of vehicle (1% Tween 80) was heated in a water bath to at least 75°C and then combined with the molten behenyl alcohol under continuous magnetic stirring, to a concentration of 20% behenyl alcohol. The resulting suspension was slowly cooled, with homogenization to a temperature of below 60°C, and then further cooled in a water bath to a temperature of 30°C. The 20% suspension was prepared once each week. Lower concentrations were prepared on the day of use by dilution of the 20% suspension with 1% w/w aqueous Tween 80.

VEHICLE
- aqueous Tween 80
- Amount of vehicle (if gavage): 1%

Animals received the test material or vehicle control formulations by gavage, at a volume-dosage of 5 mL/kg bw, using an 8 or 10 choke rubber catheter. The volume administered was calculated from body weights measured immediately before each administration.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

7 days a week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 male and 20 female per dose (main study)
10 male and 10 female per dose (toxicokinetic study)

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prestudy, weekly for the duration of the study, and at necropsy

FOOD CONSUMPTION
- calculated weekly throughout the treatment periods

FOOD EFFICIENCY:
- Weekly group mean food conversion efficiencies were calculated for the first 14 weeks of the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prestudy, and during weeks 12 and 25 of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during weeks 14 and 26
- Anaesthetic used for blood collection: Yes, anesthetized with halothane/nitrousoxide at time of sampling
- Animals fasted: Yes
- How many animals: 10 rats/sex/dose
- Parameters checked: hemoglobin concentrations, erythrocyte count, total and differential leukocyte count, platelet count, mean cell hemoglobin concentration, mean cell hemoglobin, morphology and unusual cell types, including normoblasts, anticoagulant and prothrombin time was measured
Blood samples were obtained from 6 (3 males and 3 females) nonfasted satellite rats on Day 1 and during Weeks 13 and 26 at 0.5, 1, 2, 4, 8, and 24 h after dosing

CLINICAL CHEMISTRY: Yes
- Parameters checked: alkaline phosphatase activity, alanine amino-transferase, aspartate amino-transferase,gamma-glutamyl transpeptidase, glucose, total bilirubin, total cholesterol, urea, total triglyceride, total protein,and electrolyte levels: Na, K, Cl, and C, inorganic phosphorus and electrophorectic protein, creatine

URINALYSIS: Yes
- Time schedule for collection of urine: during weeks 12 and 25
- Metabolism cages used for collection of urine: No
- Animals fasted: nonfasted waterdeprived
- Parameters checked: urine pH and protein, glucose, ketones, bilirubin, urobilinogen, blood, urine-specific gravity, the appearance and volume of urine

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- detailed examination of the external features and orifices, the neck and associated tissues, and the cranial, thoracic, abdominal, and pelvic cavities, and their viscera

HISTOPATHOLOGY: Yes
- adrenals, brain, eyes and optic nerve, femur, heart, kidneys, liver, lungs, seminal vesicles, spinal cord, stomach, thyroid, and uterus

Statistics:

For organ weights and body weight changes, homogeneity of variance was evaluated by Bartlett’s test. Whenever this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons; otherwise, a Dunnett’s test was used. Fisher’s exact test was applied as a two-tailed test, where appropriate, to the distribution of macroscopic or microscopic pathological entities. Unless stated, group mean values or incidences for the treated groups were not significantly different from those for the controls (P>0.05). The significance of intergroup differences in hematology (excluding the incidence of morphological abnormalities evident on the blood smears for rats), blood chemistry, and urinalysis (volume, pH, and specific gravity only) was assessed by Student’s t test using a pooled error variance.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male receiving 100 mg/kg/day died during Week 25.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

Toxikokinetic study:
Blood sample results were obtained from satellite animals on Day 1, and during Weeks 13 and 26 of the study. The maximum mean plasma concentration (Cmax) occurred 1 h after dosing in all males and in the majority of the females. During Week 13, females in the 100 and 1000 mg/kg bw/day dose level groups reached the Cmax at 0.5 h after dosing. 24 hours after dosing, plasma concentrations of behenyl alcohol in animals from the 10 and 100 mg/ kg bw/day dose group were generally below the limit of quantification (<10 ng/ml). In animals treated with 1000 mg/kg bw/day, mean plasma concentrations of behenyl alcohol were quantifiable on each sampling day, and these values ranged from 203.68 to 528.82 ng/mL throughout the duration of the study.
Area under the curve (AUC24) values ranged from 1043.8 to 3196.0 ng x h/mL in males and females treated with 1000 mg/kg bw/day during the study. Generally, no statistically significant sex-related differences occurred in any of the dose groups. However, statistically significant differences (P=0.040) in AUC24 values, 24 h after dosing, were observed between males and females treated with 10 and 1000 mg/kg bw/day on Day 1 and during Week 13 of the study, respectively. On Day 1, AUC24 values of 96.4 and 154.8 ng x h/mL were obtained in male and females treated with 10 mg/kg bw/day, respectively, while AUC24 values of 2797.0 and 1043.8 ng x h/ml were obtained in males and females treated with 1000 mg/ kg bw/day, respectively. The rate and extent of systemic exposure of rats to behenyl alcohol, characterized by the Cmax and AUC24, respectively, on Day 1 and during Weeks 13 and 26 of the study increased with increasing dose level; however, these increases were less than the proportionate dose increment, and there was statistically significant evidence of non-proportionality on each of the sampling days (P=0.056).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Critical effects observed:

not specified

Table 1: Maximum Mean Plasma concentrations of Behenyl Alcohol in rats and the areas under the mean plasma behenyl alcohol concentration time curves estimated up to 24 h postdose on Day 1 and during weeks 13 and 26 of the study

	Day 1		Week 13		Week 26
Dose level (mg/kg bw/day)	Males	Females	Males	Females	Males	Females
	Maximum mean plasma concentration (Cmax) (ng/mL)
10	31.16 (11.94)	49.00 (12.26)	53.28 (23.68)	42.66 (17 .44)	93.08 (49.06)	46.13 (23.26)
100	179.96 (109.07)	117.63 (25.88)	306.92 (207.29)	160.25 (83.92)	153.14 (34.42)	151.76 (40.43)
1000	292.74 (47.53)	203.68 (30.43)	525.72 (232.90)	414.91 (82.85)	526.40 (160.22)	528.82 (171.70)
	Area under the curve (AUC24)(ng x h/mL)
10	96.4 (21.0)	154.8 (19.2)	271.5 (64.6)	257.7 (71.4)	290.0 (73.0)	127.8 (55.7)
100	766.6 (89.7)	720.1 (161.0)	731.7 (180.6)	345.2 (106.9)	1072.7 (372.2)	378.9 (70.5)
1000	1858.0 (253.0)	1296.7 (186.6)	2797.0 (539.7)	1043.8 (102.9)	3196.0 (433.7)	2254.9 (442.7)

Numbers in parentheses represent standard deviations and standard errors of the Cmax and AUC24, respectively.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-Oct-2004 to 06-Dec-2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number: 88 rats (44 males and 44 females)
- Source: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®); Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: males were 8 weeks old and females were 10 weeks old
- Weight at study initiation: males: mean body weight of 317 g (range: 296 g to 345 g); females mean body weight of 223 g (range: 199 g to 261 g)
- Housing: The animals were individually housed (except during mating) in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metallic tray containing autoclaved sawdust (SISCA, Alfortville, France) was placed under these cages. During mating, gestation and lactation, the animals were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France), with wood shaving as nesting material.
- Diet: ad libitum (SNIFF R/M-H pelleted maintenance diet, batch No. 2764132 (SNIFF Spezialdiäten GmbH, Soest, Germany) distributed weekly)
- Water: ad libitum (tap water (filtered with a 0.22 μm filter)
- Acclimation period: 7 days before the beginning of the treatment period
- Allocation to group: during the acclimation period, the required number  of animals were selected according to body weight and/or clinical 
condition and allocated by sex to the groups, using a stratification procedure based on body weight (these data are not presented in the report). Body weights of the animals assigned to the study at the start of the  treatment period were within 20% of the mean weight for each sex. Identification: each animal was individually identified by an ear tattoo detailing a unique  CIT identity number.
- Contaminant analyses: The batches of diet and sawdust were analyzed by the suppliers for composition and contaminant levels. Bacterial and 
chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible  contaminants 
(sawdust: pesticides and heavy metals; diet and water:  pesticides, heavy metals and nitrosamines). No contaminants were present in the diet, drinking water or sawdust at  levels which could be expected to interfere with or prejudice the outcome  of the study. A copy of the appropriate analyses was maintained with the study records.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item dosage formulations were prepared by suspending Lauryl Methacrylate (Lauryl MA; CAS: 142-90-5) in corn oil to achieve the concentrations of 20, 60 and 200 mg/mL and were stored at +4°C, protected  from light, for up to 9 days prior to use.
- Administration: The dosage formulations were administered by gavage using a plastic syringe fitted with a metal gavage tube (length of gavage tube: 7.6 cm),  once a day, at approximately the same time. The quantity of dosage formulation administered to each animal was adjusted according to the  most recently recorded body weight with the exception that body weights on day 14 post-coitum were used to calculate individual dosages for the  pregnant females from day 14 post-coitum through parturition (day 1 post-partum) to  avoid overdosing the dams because weight gain from GD 14 to 20 (GD: gestation day) is fetal weight. A constant dosage-volume of  5 mL/kg/day was used. Control animals (group 1) received the vehicle alone. The dosage formulations were stirred continuously  throughout the dosing procedure.
VEHICLE
- corn oil
- Lot/batch no. (if required): 122K0131 and 103K0107, supplied by Sigma (Saint-Quentin-Fallavier, France)
- Purity: no data, commercial product

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity:
Two dosage formulations were prepared at 5 and 300 mg/mL of test item in corn oil to cover all the concentrations intended for use in this study. From both dosage formulations, duplicate samples were taken at three different levels of the container (top, middle and bottom) and analyzed  for concentration of the test item to evaluate the homogeneity.
- Stability: The two dosage formulations prepared for homogeneity analysis were sampled after 0 (just after preparation, homogeneity), 4 and 9 days  storage at +4°C (protected from light). The aliquots sampled on day 4  were stored frozen at -20°C pending analysis on the last sampling  occasion (day 9) when all samples were assayed. The mean concentration measured on day 0 (homogeneity) was taken as the initial value for the stability test.
- Concentration: The concentration of the test item in samples taken from each dosage formulation (including the control) prepared for use in weeks 1, 2, 4 and  8 was determined. 

Duration of treatment / exposure:

Each animal was given the appropriate dosage formulation once a day, at  approximately the same time each day, 7 days a week, according to the following schedule:
in the males: 15 days before mating, during the mating and post-mating periods until sacrifice (approximately 6 weeks in total),
in the females: 15 days before mating, during the mating period, during pregnancy and lactation, until day 5 post-partum inclusive (or until sacrifice, for un-mated females). Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

once a day, 7 days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following  the results of a previously conducted study CIT/Study No. 28196 TSR in which rats administered Lauryl MA at 0, 100, 300 or 1000 mg/kg/day for 10 consecutive days by gavage did not show any toxic effect. The highest dose-level, 1000 mg/kg/day, is the highest dose required by the guidelines.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

MORBIDITY AND MORTALITY:
- Time schedule: Each animal was checked at least twice a day for mortality and signs of morbidity (except during the acclimation period when they were checked at least once a day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed at least once a day at approximately the same time (i.e. after dosing the animals in the morning), for the recording of clinical signs. Animals were also observed in the afternoon as part of the mortality check and any clinical signs were recorded.

All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal's treatment, before
the first day of treatment and then once a week thereafter.The animals were randomized in order to ensure "blind" evaluation, except for examination performed before the first day of treatment.
The following parameters were assessed:
- "touch escape" or ease of removal from the cage,
- in the hand: fur appearance, chromodacryorrhea, chromorhinorrhea, salivation, lachrymation, piloerection, eye, exophthalmia, mucous membrane, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy behaviour and breathing, ataxia, hypotonia.

Reactivity to manipulation or to different stimuli (FOB):
In five males and five females per group, the examinations listed below were conducted shortly before terminal sacrifice, and before blood sampling for clinical pathology. The observer performing the evaluation was not aware of the treatment group of the animal. The animals were randomly selected in order to ensure "blind" evaluation.
The following measurements, reflexes and responses were recorded:
. touch response,
. forelimb grip strength qualitative approach,
. pupil reflex,
. visual stimulus,
. auditory startle reflex,
. tail pinch response,
. landing foot splay,
. righting reflex,
. at the end of observation: rectal temperature.

Motor activity
Motor activity was measured in five males and five females using automated infra-red sensor equipment, recording individual animal activity over a
one-hour period. The females were evaluated on or near day 17 post-coitum to avoid removal of the dam from her pups after parturition and the
males were evaluated at approximately the same time as the females and near the time of sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7,14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption/Compound intake: Yes
The quantity of food consumed by each animal was recorded once a week, over a 7-day period, from the first day of treatment through gestation (days 0-7, 7-10, 10-14, 14-17 and 17-20 post-coitum intervals) and lactation (days 1-5 post-partum interval). During the mating period, the food consumption was not recorded for males or females. Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper at the end of the specified interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Animals fasted: Yes
- How many animals: 10; five males and five females (adults) from each group at terminal sacrifice
- Parameters checked: Lithium heparin tubes: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS),
Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G),
Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT),
Tubes without anticoagulant: Biles acids (BIL.AC)

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
- How many animals: 10; five males and five females (adults) from each group at terminal sacrifice
- Parameters checked: EDTA tubes: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin
concentration (MCHC), Mean cell hemoglobin (MCH) Thrombocytes (PLAT), Leucocytes (WBC), Differential white cell count with cell morphology
. neutrophils (N)
. eosinophils (E)
. basophils (B)
. lymphocytes (L)
. monocytes (M)
Sodium citrate tubes: Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB)

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 5; first surviving five males (adults) from each group at terminal sacrifice
- Parameters checked: Quantitative parameters: Volume (VOLUME), pH (pH), Specific gravity (SP.GRAV)
Semi-quantitative parameters: Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (BLOOD), Urobilinogen (UROB)
Cytology of sediment Microscopic:
. Leucocytes (WBC)
. Erythrocytes (RBC)
. Cylinders (CYLIN)
. Magnesium ammonium phosphate crystals (AMM.PH)
. Calcium phosphate crystals (CAL.PH)
. Calcium oxalate crystals (CAL.OX.)
. Cells (CELLS)
Qualitative parameters: Appearance (APP), Color (COLOR)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Motor activity
The statistical comparison of results of motor activity (horizontal and rearing movements recorded from 5 males and 5 females per group) was
performed, according to the following steps:
. step 1: normality and homogeneity of variances (Kolmogorov-Smirnov and Bartlett test respectively),
. step 2: parametric (ANOVA plus Dunnett test if necessary) or non-parametric (Kruskal-Wallis plus Dunn test if necessary) tests were conducted
based on the results obtained at step 1.
These statistics were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all
tests.The other data are compared by one-way analysis of variances (ANOVA) and Dunnett test (quantitative values) or by Fisher exact probability test
(proportions).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hypersalivation in males and females dosed 300 or 1000 mg/kg bw/day during premating (for males) and furthermore during gestation and lactation (for females).

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Females given 1000 mg/kg bw/day gained 18% less weight than the controls between day 0 and day 7 post-coitum. This weight difference was statistically insignificant (limited to the first week of gestation). No other treatment-related effects on body weight were noted.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males:
The food consumption of treated males was similar to that of the controls during the study.
Females:
There was no effect of treatment on group mean food consumption during the premating or lactation periods.
Between day 7 and day 10 post-coitum, all groups given Lauryl MA consumed less food than the controls, with no dose-relationship trend, achieving statistical significance at 100 and 1000 mg/kg bw/day (-17%, p<0.05 and -25%, p<0.001, respectively). Due to the lack of dose response and lack of consistency with other time periods in the study, these differences in food consumption were not considered of toxicological importance.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related effect on any of the hematological parameters examined.
When compared with the mean control values, a lower value in total white cell count was recorded for females in the 1000 mg/kg bw/day group (4.95 vs 7.34 g/L: -33%, not statistically significant). This change was not considered related to treatment with the test item because of the great variability and the great inter-individual variations of this parameter and the lack of effects on other related parameters (differential cell counts).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was an increase in blood glucose concentration in male rats given 1000 mg/kg bw/day (7.63 mmol/L, p<0.01) when compared with the controls (5.78 mmol/L); a relationship to the treatment with the test item cannot be excluded. However, this variation was not considered to be of toxicological importance since the number of animals/group was limited to five, all values were within historical data range (mean: 6.69 mmol/L, [5.22-9.04 mmol/L]) and there were no histopathological findings in the liver.
All other differences were considered not to be relevant since either no clear dose-relationship was evident (increased triglyceride and cholesterol plasma concentration in test item-treated females), or differences were slight (decrease in plasmatic protein concentration and increase in liver enzyme activities in females).

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on pH, specific gravity or volume of urine produced by the males in any group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Motor activity:
There were no differences in the measured motor activity (horizontal and rearing movements) which could be attributed to treatment with the test item. Detailed clinical observations and reactivity to manipulation or different stimuli (FOB). There was no evidence of disturbance of either autonomal or physiological functions at any dose-level.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on absolute or relative organ weights in any dose groups.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mating at any dose-level. The male and female fertility indices were unaffected by treatment; all mated females, except one given 1000 mg/kg bw/day, were pregnant with live fetuses. The duration of gestation was similar between the control and test item-treated groups. There was no effect of treatment on the mean number of liveborn pups or on pup death after birth. There were no external pup abnormalities in the control or test item-treated groups. There was no effect of treatment on mean pup body weight or body weight gain for males or females. The sex ratios on days 1 and 5 post-partum were similar in the control and test item-treated groups. No relevant findings were observed in the pups sacrificed on day 6 post-partum.

Key result

Dose descriptor:

NOAEL

Remarks:

(parental toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOEL

Remarks:

(toxic effect, reproductive performance and on developmental toxicity F1)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD TG 422 (Read Across)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

Due to the very low vapour pressure, which is estimated as 1.96E-5 Pa at 25°C for Docosyl methacrylate, it is extremely unlikely that inhalation toxic concentrations could ever be reached. Hence, the inhalation pathway is not considered a relevant route of exposure for long-chain methacrylate esters (C12 – C22) including Docosyl methacrylate as solid waxen substance.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Study duration:

chronic

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

oral

No studies on repeated oral dose toxicity for the target substance Docosyl methacrylate are available. Therefore, read-across from studies with the structurally analogous substance Dodecyl methacrylate (CAS 142-90-5) and the primary metabolite Docosan-1-ol (Behenyl alcohol, CAS 661-19-8) was performed.

In two subchronic toxicity studies, CD rats and beagle dogs were administered Docosan-1-ol (Behenyl alcohol) by oral gavage for at least 26 weeks at doses of 0, 10, 100, or 1000 mg/kg bw/day for rats and 0, 20, 200, or 2000 mg/kg bw/day for dogs. No clinical signs of systemic toxicity, no effects on body weight gain, organ weight, food consumption, hematology, and clinical biochemistry were observed in rats. Gross pathology and histopathology examinations revealed no treated-related findings. Compound-related effects in dogs were limited to observations of pale feces, indicative of unabsorbed Docosan-1-ol, at doses of 2000 mg/kg bw/day. No histopathological changes were observed in dogs dosed with Docosan-1-ol. The NOAEL was 1000 mg/kg bw/day for rats and 2000 mg/kg bw/day for dogs, the highest doses tested (Iglesias et al. 2001).                                        

In a study according OECD TG 422 Dodecyl methacrylate was administered to male and female Sprague-Dawley rats by oral gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day. There were no premature deaths observed during the study. At 1000 mg/kg bw/day, hypersalivation was recorded in males and females. Lower body weight gain was recorded in females during the GD 0-7 interval at the highest dose level, which was statistically insignificant and limited to the first week of gestation. Increased plasma glucose concentrations were recorded in males at the highest dose level. This variation was not considered to be of toxicological importance since the number of animals/group was limited to five, all values were within historical data range and there were no histopathological findings in the liver. At 300 mg/kg bw/day, a few animals had hypersalivation. At 100 mg/kg bw/day, no treatment-related effects were detected. Hypersalivation was not considered to be a sign of toxicity. There were no treatment-related effects on absolute or relative organ weights, urinalysis or behavior in any dose groups. No substance-induced effects on male and female reproductive performance or on the progeny of the parental rats at any dose-level were observed. There were no treatment-related findings at histopathological examination. Based on the experimental conditions of this study, the NOAEL for parental toxicity was considered to be 1000 mg/kg bw/day and the NOEL for toxic effect on reproductive performance and on developmental toxicity was greater than or equal to 1000 mg/kg bw/day (SAM UNTER 05 -044).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Overall, in consideration of all data available for structurally analogous methacrylate esters and the primary metabolites of the target substance, there is no convincing evidence of repeated dose oral toxicity. As a result the substance is not considered to be classified for repeated oral dose toxicity, as amended for the tenth time in Regulation (EU) No 2017/776.