Cyclohexyl methacrylate

Endpoint summary

Description of key information

oral

registered substance cyclohexylmethacrylate (CAS# 101-43-9): ca. 100 -day oral gavage study (OECD 422/OECD 408), rat: NOAEL = 300 mg/kg bw/d (BASF SE, 2018)

registered substance cyclohexylmethacrylate (CAS# 101-43-9): 28-day oral gavage study (OECD 407 ), rat: NOAEL = 300 mg/kg bw/d (BASF SE, 2010)

analogous substance cyclohexanone (CAS# 108-94-1): 90-day drinking water study (OECD 408), rat: NOAEL = 143 mg/kg bw/d (BASF SE, 1994)

analogous substance cyclohexanone (CAS# 108-94-1): 2-y drinking water study (equivalent to OECD 453), rat: NOAEL = 462 mg/kg bw/d (Lijinsky et al., 1986)

analogous substance methylmethacrylate (CAS# 80-62-6): 2-y drinking water study, rat: NOAEL of >= 90.3 mg/kg bw/day and 193.8 mg/kg bw/day, for males and females, respectively (Borzelleca et al., 1964)

 

inhalation

hydrolysis product cyclohexanol (CAS# 108-93-0), inhalation screening study, rat: NOAEC maternal toxicity 610 mg/m³; NOAEC fertility = 1640 mg/m³ (US EPA, 2010)

hydrolysis product methacrylic acid (CAS# 79-41-4): 90-day whole body inhalation study (OECD 413), rat: NOAEC systemic = 352 mg/m³; LOAEC local =  (BASF SE, MAA Task Force, 2008)

analogous substance n-butylmethacrylate (CAS# 97-88-1): 28/14-d whole body inhalation study (OECD 412), rat: NOAEC systemic = 11175 mg/m³; NOAEC local = 1832 mg/m³ (Methacrylate Producers Association, Hagan et al., 1993)

analogous substance methylmethacrylate (CAS# 80-62-6): 90-day inhalation study (equivalent to OECD 413), mouse: NOAEC systemic = 12300 mg/m³; NOAEC local = 4100 mg/m³ (Rohm & Haas, 1980)

analogous substance methylmethacrylate (CAS# 80-62-6): 90-day inhalation study (equivalent to OECD 413), rat: NOAEC systemic = 2050 and 4100 mg/m³ for male and female rats, respectively (Rohm & Haas, 1980)

analogous substance methylmethacrylate (CAS# 80-62-6): 2-y whole body inhalation study (equivalent to OECD 453), rat: NOAEC systemic = 1640 mg/m³; NOAEC local = 104 mg/m³ (Lomax et al., 1997)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-24 to 2017-10-17

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:

29 Jul 2016

Deviations:

yes

Remarks:

Please refer to Principles of Method.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 Sep 1998

Deviations:

yes

Remarks:

Please refer to Principles of Method.

Principles of method if other than guideline:

The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.
In addition groups of 10 males and 10 females, selected from F1 pups to become F1 rearing animals, were treated with the test substance at doses of 0, 100, 300 and 1000 mg/kg bw/d postweaning until puberty.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 010545EDA0
- Expiration date of the lot/batch: Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, avoid temperatures > 35 °C

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 36 +/- 1 days
- Weight at study initiation: males: 118.4-142.6 g, females: 92.7-124.3 g
- Fasting period before study: no
- Housing: as groups of 4 in Polysulfonate cages supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany with exceptions: during overnight matings, male and female mating partners were housed together in Polycarbonate cages type III (same supplier) and pregnant animals and their litters were housed together until PND 21 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne
GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, drinking water
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and for microbiological contaminants. The diet was found to be suitable. The concentration of microorganisms did not exceed 1*10^5/g feed.
The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE. The drinking water was found to be suitable.
The bedding and the enrichment is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals) by the producer. It was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Vehicle:

water

Remarks:

drinking water (with 10 mg/ 100 mL Cremophor EL)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water with 10 mg/100 mL Cremophor EL and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 1, 3 and 10 g/100 mL
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in drinking water + 10 mg/100 mL Cremophor EL for a period of 7 days at room temperature were carried out prior to the start of the study.
At the beginning and at the end of premating, once during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. Of each sample, one additional reserve sample was retained.
The samples collected at the beginning of the administration period and during lactation period were analyzed. The remaining samples were stored at -20 °C.

Results:
The stability of test substance in drinking water + 10 mg/100 mL Cremophor EL was demonstrated for a period of 7 days at room temperature.
The homogeneous distribution of the test substance in drinking water + 10 mg/100 mL Cremophor EL was demonstrated.
Measured values for the test item were in the expected range of the target concentrations (90 - 110 %), demonstrating the correctness of the preparations. Measured values of samples 30 - 32 were slightly under the specification limit of 90 % (average 88.7 %), however, the re-check using the retain samples 30R-32R proved to be within the specification limits of 90-110 % (average 92.2 %).

Duration of treatment / exposure:

Animals of parental generation were treated for:
females: 126/133 days
males: 109/110 days

The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females.

Frequency of treatment:

once daily, 7 days per week, exception: no administration to animals being in labor

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:

12 parental animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on request of the sponsor.

Positive control:

No positive control conducted.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the administration period all animals were checked daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals once prior to the first administration (day 0) and at weekly intervals thereafter.
- Detailed clinical observations checked in table No.1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined once a week for male and female parental animals. Food consumption was not determined after the 10th premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20. Food consumption of females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Anaesthetic used for blood collection: Yes, isoflurane
- How many animals: in the first 10 surviving parental males (fasted) per group and a maximum of 10 females (fasted) with litters (in order of delivery) per group
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: at termination, females: at PND 14
- Animals fasted: Yes
- How many animals: in the first 10 surviving parental males per group and a maximum of 10 females with litters (in order of delivery) per group
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes, as part of detailed clinical observations.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: end of administration period
- Dose groups that were examined: all, first 5 parental males and the first 5 females with litter (in order of delivery) per group
- Battery of functions tested are checked into table No. 2-4. In addition motor activity was determined in the dark using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany) with 18 infrared beams per cage. The numbers of beam interrupts were counted over 12 intervals, each lasting 5 minutes.

IMMUNOLOGY: No

OTHER: Thyroid hormones
Blood samples for T4 and TSH were collected from retrobulbar venous plexus under isoflurane anesthesia.
- Time schedule for collection: from all dams at PND 14 and all males at termination, animals were fastened
- Dose groups that were examined: all males

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: Were determined as listed in table No. 7.

HISTOPATHOLOGY: Yes. The organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution as listed in table No. 8. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.

Other examinations:

Furthermore observations and examinations concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups were conducted. The details on those determinations are provided in IUCLID section 7.8.1.

Statistics:

Means and standard deviations were calculated.
Further statistics were performed as listed in table No. 9.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All male and all female animals of the mid- and high-dose groups (300 and 1000 mg/kg bw/d) showed salivation at least on one occasion during the treatment period. Individual males (three) of the low-dose group occasionally showed salivation during the mating and postmating periods. Due to salivation several male and female animals of the high- and mid-dose groups showed red discolored fur in the mouth or nose region during the treatment.
The temporary salivation and discolored fur was considered to be test substance-induced. From the temporary, short appearance of salivation immediately after dosing it is likely, that these findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups (01 - 03 , 100, 300 and 1000 mg/kg bw/d) during the study.
There were sporadic findings in individual F0 animals as follows:
One high-dose male animal (No. 43) showed protruding eyeball during mating days 4 - 14 and postmating days 0 - 22. One mid-dose male animal (No. 27) showed hyperthermia and sparse fur (grade: severe) during mating days 1 - 14 and during postmating days 0 - 2 and 0 - 21, respectively. One sperm positive high-dose female (No. 138) and one sperm negative middose female (No. 136) did not deliver F1 pups. None of these findings is considered to be treatment-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related mortalities in any of the groups.
One F0 female of test group 03 (1000 mg/kg bw/d) showed piloerection and vaginal discharge (light yellow) on GD 19 and was found dead on GD 20. Histopathology revealed a marked inflammation of the placenta. This was considerd to be a spontaneous finding.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control during the entire study period.
The statistically significantly lower body weight change in the mid-dose males during premating days 63 – 70, the statistically significantly higher body weight change in the mid-dose females during premating days 38 - 35 as well as significantly higher body weight change in the mid and high-dose females during PND 0 - 21 were considered as spontaneous in nature and not as treatment related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all test substance-treated F0 male and female animals (100, 300 and 1000 mg/kg bw/d) was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related findings were observed.
One high-dose male animal (No. 43) showed protruding eyeball during mating days 4 - 14 and postmating days 0 - 22. In the pupillary reflex test one out of 5 examined male animals of dose group 01 and two out of 5 examined male animals of dose group 03 showed a retarded adaption of the pupil to light. These findings were regarded as spontaneous findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in males of test group 03 (1000 mg/kg bw/d) red blood cell (RBC) counts and hematocrit values were slightly, but significantly decreased.
Additionally, in males of test group 03 (1000 mg/kg bw/d) absolute eosinophil cell counts were slightly lower compared to controls, but this was the only changed differential blood cell fraction and therefore this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). In females of test group 03 (1000 mg/kg bw/d) at lactation day 14, absolute and relative neutrophil counts were significantly lower and relative lymphocyte counts were significantly higher compared to controls. No changes among the total white blood cell (WBC) counts and other hematology parameters occurred in these individuals. Therefore, these alterations were regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).
In males of test groups 02 and 03 (300 and 1000 mg/kg bw/d) after the administration period, prothrombin time (Hepatoquick’s test, HQT) was significantly reduced, but the values were within the historical control range (males, HQT 34.6-40.2 sec). Therefore, this change was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After the administration period in males of test group 03 (1000 mg/kg bw/d), total protein and globulin values as well as cholesterol and potassium values were significantly increased.
After lactation day 14 in females of test group 03 (1000 mg/kg bw/d) also total protein and globulin levels as well as albumin values were significantly higher compared to controls. Additionally, in females of test group 03 (1000 mg/kg bw/d) creatinine values were significantly lower compared to controls.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among urinalysis parameters were observed.
After the administration period in males of test groups 02 and 03 (300 and 1000 mg/kg bw/d), urine pH values were significantly decreased whereas urine specific gravity was significantly increased. Additionally, in males of test group 03 (1000 mg/kg bw/d) incidences of ketone bodies were significantly higher compared to controls. No hint of a dysregulation of the energy metabolism (e.g. changes in serum glucose, creatinine, triglycerides) was observed among these individuals. It can be assumed, that acidic metabolites of the compound are excreted via the urine decreasing the urine pH and maybe increasing the urine specific gravity. Methacrylic acid as metabolite of the compound is further degraded to Acetyl-CoA, and high abundance of this molecule results in formation of acetoacetate as ketone body, which is excreted via the urine (Greim et al., 1995). These changes are significant only in males, because metabolism rate is normally higher in this sex. Therefore, the mentioned altered urine parameters in males of test groups 02 and 03 are regarded as treatment-related, but not as adverse.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

The open field observations did not reveal any test substance-related findings in F0 male and female animals of test groups 01 and 02.
Three out of 5 examined male animals of dose group 03 (1000 mg/kg bw/d) showed slight salivation (area around the mouth was moist), which was considered to be treatment-related.

There were no test substance-related findings in male and female F0 animals of all test groups in sensorimotor tests/reflexes.
In the pupillary reflex test one out of 5 examined male animals of dose group 01 and two out of 5 examined male animals of dose group 03 showed a retarded adaption of the pupil to light. One out of 5 examined male animals of dose groups 02 and 03, respectively, showed very frequent vocalizations when touched. These were regarded as spontaneous findings.

No test substance-related impaired quantitative parameters were observed in male and female animals of all test groups.
As there was no dose-response the statistically significantly lower values of grip strength of forelimbs in females of dose group 01 was considered as spontaneous in nature and not treatment-related.

No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights:
Mean absolute weights significantly increased are shown in table 10.
The terminal body weight of male animals was slightly decreased without statistical significance in test group 02 (by 4 %) and test group 03 (by 7 %).
All other mean absolute weight parameters did not show significant differences when compared to the control group 00.

Relative weights
Mean relative organ weights significantly increased or decreased are shown in table 11.
All other mean relative weight parameters did not show significant differences when compared to the control group 00.
The increase in absolute and relative liver and kidney weights in test group 03 male and female animals was considered to be treatment-related, but the kidney weight changes were considered as non-adverse.
The increase in relative adrenal and pituitary gland weights in test group 03 males and relative liver weights in test group 02 males was regarded as incidental and possibly secondary to the decreased terminal body weights in these groups. Additionally, weights were within historical controls for studies carried out according to the OECD 422 guideline (adrenal historical controls absolute/relative: 59.4-77.4 mg/ 0.016- 0.021 %, this study test group 03 absolute 65 mg, relative 0.018 %; liver historical controls relative 2.108-2.45 %, this study 2.423 %.).
Pituitary gland weights were compared with historical controls for studies carried out according to the OECD 408 guideline - as these values are not normally measured in studies carried out according to the OECD 422 guideline (pituitary gland historical controls absolute/relative: 7.8- 15.2 mg/0.002-0.004 %, this study 9.167 mg/0.003 %).
The decreased relative organ weights of the spleen in test group 01 male animals was regarded as incidental as there was no dose response and no significant histopathological changes in test group 03. The increased spleen weights of test group 03 males were due to the infiltration by lymphoma cells in the spleen of animal 41 (absolute spleen weight of animal 41: 2.94 g, while the other animals of this group showed weights around 0.6 g).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the liver in female animals of test group 03.
In female animals, minimal hepatocellular hypertrophy was noted in the liver, with incidences and grading according to the table 12.
In male animals, minimal follicular hypertrophy/hyperplasia was noted in the thyroid gland, with incidences and grading according to the table 13.

In females, the predominant pattern of hepatocellular hypertrophy was centrilobular, the animal showing the periportal pattern was the premature decedent (female animal 137), where the liver was markedly enlarged even on gross evaluation. This was the only animal examined during pregnancy, therefore this possibly confounding factor should be taken into account and the relationship to treatment of the observed periportal hypertrophy is questionable.

The findings observed in the thyroid gland in male animals of test group 03 were finally considered as secondary in nature due to the observed functional liver effects and thus, as not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One male animal of test group 03 (animal No. 41) showed a lymphoma, this was assessed as incidental, as the occurrence of lymphoma has been documented in single control animals of this age in this laboratory.

Other effects:

no effects observed

Description (incidence and severity):

Furthermore results concerning estrous cycle, male reproduction, female reproduction and delivery and litter/pups are provided in IUCLID section 7.8.1.

Thyroid hormones:
In parental males (test groups 01, 02 and 03; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table No. 10 Absolute organ weights

When compared with control group 00 (=100%), the following mean absolute weights were significantly increased in test group 03:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Terminal body weight	101 %	96 %	93 %	101 %	102 %	100 %
Liver	99 %	101 %	123 %**	101 %	108 %	122 %**
Kidney	94 %	94 %	113 %**	104 %	105 %	117 %**

*p ≤ 0.05; **p ≤ 0.01

 

Table No. 11 Relative organ weight

When compared with control group 00 (=100%), the following mean relative organ weights were significantly increased or decreased in one or more test groups:

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Adrenal glands	103 %	106 %	123 %**	-	-	-
Kidneys	93 %	97 %	120 %**	102 %	102 %	117 %**
Liver	98 %	105 %**	131 %**	100 %	105 %	122 %**
Pituitary gland	102 %	105 %	114 %**	-	-	-
Spleen	86 %*	91 %	127 %	-	-	-

*p ≤ 0.05; **p ≤ 0.01

 

Table No. 12 Histopathology

Liver	Female animals
Test group (mg/kg bw/d)	0 (control)	1 (100)	2 (300)	3 (1000)
No. of animals	12	12	12	12
Hypertrophy, centrilobular	0	0	0	3
Grade 1				3
Hypertrophy, peripheral	0	0	0	1
Grade 2				1

 

Table No. 13 Histopathology

Thyroid gland	Male animals
Test group (mg/kg bw/d)	0 (control)	1 (100)	2 (300)	3 (1000)
No. of animals	12	12	12	12
Hypertrophy/hyperplasia, follicular	0	0	0	2
Grade 1				2

 

DISCUSSION

In this Modified Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD 422/408), the test substance cyclohexyl methacrylate was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) to screen for potential repeated dose, reproductive and developmental toxicity. The informative value of the OECD 422 test was increased by a longer premating treatment period (10 weeks) and a postweaning follow-up of selected offspring until puberty.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 7 days under ambient conditions.

The majority of male and female F0 animals in test groups 3 and 2 (1000 mg/kg bw/d and 300 mg/kg bw/d) showed salivation for a time period after gavage treatment. Secondary to salivation several male and female animals of these test groups showed red discolored fur in the mouth or nose region during the treatment. From the temporary, short appearance of salivation immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. As such, this local finding was not considered to be an adverse and systemic-toxicologically relevant effect. While occasional salivation was the only notable finding during detailed clinical observations, no abnormalities were noted in FOB or motor activity measurements.

Food consumption and body weights/body weight gain remained unaffected in all treatment groups in F0 parents.

Dysregulation of the liver cell metabolism was detected in males and females treated with 1000 mg/kg bw/d, which was seen as increase of serum total protein and globulin values (males and females), increase of cholesterol and potassium levels (males) and high albumin level and low creatinine values (females). Additionally a marginal anemia was observed because of decreased red blood cell (RBC) counts and hematocrit values. Furthermore the liver showed a marked weight increase in animals of both sexes treated with 1000 mg/kg bw/d. A corresponding histopathological correlate in the form of hepatocellular centrilobular hypertrophy could only be detected in female rats. No further adverse or primary substance-related effects were detected in animals at 1000 mg/kg bw/d. At 100 and 300 mg/kg bw/d no test substance-related adverse findings were determined.

CONCLUSION

Under the conditions of the present modified OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity of cyclohexyl methacrylate was 300 mg/kg bw/d for male and female rats, based on functional liver impairment in F0 parental rats at 1000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study was conducted according to guideline and GLP.

System:

haematopoietic

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(Extention of the exposure period, additional repeated dose group for females and a 4-week recovery period (males))

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males: 16 wks (10 weeks premating)
Females: 13 wks
(post exposure observation time: 4 weeks)

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0 ppm

Dose / conc.:

50 ppm

Remarks:

0.21 mg/L nominal conc.

Dose / conc.:

150 ppm

Remarks:

0.61 mg/L nominal conc.

Dose / conc.:

450 ppm

Remarks:

After 10 weeks exposure, the level was reduced to 400 ppm due to slight mortality and the perceived additional stress of mating (females).
1.84/1.64 mg/L nominal conc.

No. of animals per sex per dose:

15 (10 animals/sex/dose in the main group; 5 animals/sex/dose in the recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Post-exposure recovery period in satellite groups: 4 weeks, 5 rats/sex/dose

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection: After 5 weeks (5 animals/sex/group), 13 weeks (10/females/group), 18 weeks (10 males/group), and after the 4-week recovery period (5 animals/sex/group) were selected for blood collection

CLINICAL CHEMISTRY: Yes
- Time schedule for collection: After 5 weeks (5 animals/sex/group), 13 weeks (10/females/group), 18 weeks (10 males/group), and after the 4-week recovery period (5 animals/sex/group) were selected for blood collection

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Complete necropsies were performed on all rats and specific organs and tissues were weighed and examined microscopically.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the weekly detailed clinical observations, no compound related effects were seen. However, in observations conducted immediately post-exposure, adverse clinical signs such as decreased activity and prostration were seen in a few animals (both sexes) in the 450/400 ppm exposure group. Please also refer to Table 1.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Single males were found dead at 450 ppm on Days 37,38 and 60 of the study. A single female rat was euthanized in extremis on Day 17. One female was found dead on Day 31. No cause of death could be determined.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Haematological findings:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No compound related effects were seen.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEC

Remarks:

(systemic toxicity)

Effect level:

0.61 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Dose descriptor:

LOAEC

Effect level:

1.64 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

no

Table 1: Mortality

	Test group 0 0 ppm (0 mg/L)	Test group 1 50 ppm# (0.21 mg/L)	Test group 2 150 ppm (0.610 mg/L)	Test group 3 450/400 ppm+ (1.84/1.64 mg/L)
Males	0/15	1/15	0/15	3/15
Females	0/15	1/15	0/15	2/15
#50 ppm group: incidentally, one male died during blood collection on Day 126 and one female was found dead on Day 32. +450/400 ppm: 3 males were found dead on Days 37, 38 and 60; 1 female was found dead on Day 31 and 1 female was euthanizedin extremison Day 17. Clinically decreased activity, prostration, and difficult breathing were seen prior to death.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

610 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The weight of evidence is considered appropriate and the quality of the data is sufficient for assessment.

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One combined OECD 422/408 oral sub-chronic toxicity study in rats using the test substance cyclohexyl methacrylate is available. In addition a sub-acute oral toxicity study with the test substance is available and used as supporting evidence. Furthermore data of the ketone (cyclohexanone) of the hydrolysis product cyclohexanol and the lower methacrylate ester methyl methacrylate are considered as supporting data.

 

Key study

Oral repeated dose toxicity

Cyclohexyl methacrylate

A “Modified Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” in rats was conducted according to OECD 422 and OECD 408 using the test substance. Cyclohexyl methacrylate was administered daily as an aqueous preparation to groups of 12 male and 12 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (drinking water + 10 mg/100 mL Cremophor EL). The duration of treatment covered a 10 weeks premating period and 2 weeks mating period in both sexes, approximately 3 weeks postmating in males, and the entire gestation period as well as 21 days of lactation and up to 15 days postweaning, or 38 days postmating for sperm negative females. Thus, the reliability regarding the possible systemic toxicity properties was increased due to the longer premating treatment period (10 weeks) and a postweaning follow-up of selected offspring until puberty.

After 10 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and thereafter at weekly intervals. Food consumption of the F0 parents and F1 rearing animals was determined once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13, 13 - 17 and 17 - 21. Body weights of F0 parents and F1 rearing animals were determined once a week.

Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

 

The stability, the homogeneous distribution and correct concentrations of the test substance in drinking water + 10 mg/100 mL Cremophor EL were demonstrated by analysis.

The majority of male and female F0 animals in test groups 3 and 2 (1000 mg/kg bw/d and 300 mg/kg bw/d) showed salivation for a time period after gavage treatment. Secondary to salivation several male and female animals of these test groups showed red discolored fur in the mouth or nose region during the treatment. From the temporary, short appearance of salivation immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. As such, this local finding was not considered to be an adverse and systemic-toxicologically relevant effect. While occasional salivation was the only notable finding during detailed clinical observations, no abnormalities were noted in FOB or motor activity measurements.

Food consumption and body weights/body weight gain remained unaffected in all treatment groups in F0 parents.

Concerning clinical pathology in F0 males and females of test group 03 (1000 mg/kg bw/d) serum total protein and globulin values were increased. Additionally, in males of this test group cholesterol and potassium levels were significantly increased and in females of test group 03 albumin levels were higher and creatinine values were lower compared to controls. The mentioned changes were considered to reflect a dysregulation of the liver cell metabolism. Additionally, in F0 males of test group 03 (1000 mg/kg bw/d) a marginal anemia was observed.

The liver showed a marked weight increase in test group 03 F0 animals of both sexes. As corresponding histopathological correlate, hepatocellular centrilobular hypertrophy could only be detected in F0 female rats of test group 03.

For parental animals of the 100 and 300 mg/kg bw/d dose groups no test substance-related adverse findings were determined. Besides these liver findings no further adverse or primarily substance-induced findings were noted.

 

Under the conditions of the present modified combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats (OECD 422/408), the NOAEL (no observed adverse effect level) for general, systemic toxicity of Cyclohexyl methacrylate was 300 mg/kg bw/d for male and female rats, based on functional liver impairment in F0 parental rats at 1000 mg/kg.

 

Supporting studies

Oral repeated dose toxicity

Cyclohexyl methacrylate

In a supporting sub-acute oral toxicity according to OECD guideline 407, cyclohexyl methacrylate was administered to 5 Wistar rats/sex/dose at concentrations of 0, 100, 300 and 1000 mg/kg bw/d per gavage for a total of 28 days.

Regarding clinical examinations, signs of general systemic toxicity were not observed up to a dose level of 1000 mg/kg bw/d. Salivation was seen after dosing in all rats of test group 3 (1000 mg/kg bw/d) as well as in 4 male and 2 female animals of test group 2 (300 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Among clinical pathology parameters some electrolyte and magnesium serum levels were altered in rats of both sexes of test group 3 (1000 mg/kg bw/d), which might be treatment-related, although a patho-physiological reason could not be found. In males of the same test group the globulin values and, therefore, the total protein levels were increased. Because no indication of an inflammatory process was found, it is highly probable that transport globulins were synthesized in consequence of the administration of the compound. Higher urea levels in females of test group 3 (1000 mg/kg bw/d) in the absence of any other altered renal parameter indicate an increased protein metabolism. Regarding pathology, target organs were the kidneys and the liver. In the kidneys, eosinophilic droplets were observed in the cytoplasm of proximal tubules in all male animals. The severity was increased in a dose-related manner in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d). Eosinophilic droplets in the proximal convoluted tubules most likely represent alpha 2µ globuline, a poorly hydrolyzable, low molecular weight protein characteristic for male rats. The increase of eosinophilic droplets was regarded to be treatment-related but since no tubular injury was found in this study, the increase of eosinophilic droplets was regarded as non-adverse. The increase of eosinophilic droplets could not explain the increased kidney weights that were observed in males of test group 2 (300 mg/kg bw/d) as well as in males and females of test group 3 (1000 mg/kg bw/d). The increase of kidney weights was related to treatment but was regarded to be non-adverse. Although there were no histopathological correlates for the significantly increased absolute and relative liver weights in males and females of test group 3 (1000 mg/kg bw/d), a treatment-related effect was assumed but was assessed as adaptive.

In conclusion, with regard to clinical pathology parameters the oral administration of Cyclohexyl methacrylate by gavage over a period of 4 weeks revealed signs of toxicity in male and female Wistar rats at dose levels of 1000 mg/kg bw/d. Salivation was considered to be related to either the bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 300 mg/kg bw/d for male and female Wistar rats.

 

Cyclohexanone (sub-chronic repeated dose toxicity study)

A sub-chronic toxicity study with cyclohexanone according to OECD guideline 408 is available (BASF SE, 1994). Cyclohexanone as ketone is fastly reduced to the corresponding alcohol (cyclohexanol) in the body (MAK Collection, Cyclohexanone, 1992)

Cyclohexanone was administered to 10 Wistar rats/sex/dose at concentrations of 0, 500, 2000 and 7000 ppm (about 40, 143 and 407 mg/kg bw) via drinking water for a total of 90 days.

No signs of toxicity were observed during clinical examination or ophthalmoscopy, respectively. Statistically significant decreases in water consumption occurred sporadically in test group 1, more often in test group 2 and over the whole study period in test group 3. In test group 3 the overall water consumption of males was reduced about 31 % and that of females about 37 %. Food consumption was not influenced in test group 1 and male test group 2 animals. It was sporadically significantly reduced in female animals of group 2 and 3, but nearly over the whole administration period in males of test group 3. These findings were attributed to the strong odor and taste the test substance, preventing full acceptance of the treated water and the food by the animals. The major treatment-related effect noted in the clinical pathology testing was the increase in total cholesterol in the serum of both sexes, which is probably due to a slight impairment of lipid metabolism at the high concentration of 7000 ppm. The increase in serum proteins seen in the high-dose animals might be related to the reduced water consumption. However, the increase in platelets in the females is considered of minor toxicological importance, because the significance of this isolated finding is not obvious. In conclusion, the results of the clinical chemistry and hematology examinations revealed some treatment-related effects in the highest dose group only which are probably a result of slight changes in lipid metabolism or of reduced water consumption. The only substance related patho-morphological effect was the decreased terminal body weight in the high dosage group. All other findings were judged to be of spontaneous or incidental nature.

Conclusively, at the concentration of 7000 ppm signs of toxicity were observed. The reduction of water and food consumption at the lower concentrations is not considered to be an adverse health effect, but caused by the strong odor and taste of the test substance. Therefore, the NOAEL for this drinking water study is 2000 ppm, corresponding to a dose of 143 mg/kg body weight.

 

Cyclohexanone (chronic repeated dose toxicity study)

A chronic toxicity study with cyclohexanone over a period of 2 years equivalent to OECD guideline 453 is also available (Lijinsky et al., 1986). 52 F344 rats/sex/dose were continuously treated at concentrations of 3300 and 6500 ppm (corresponds to 462 and 910 mg/kg bw /d) via drinking water.

Both male and female rats receiving 6500 ppm of cyclohexanone for 2 years showed a considerably smaller weight gain throughout the experiment compared with the weight gain seen in controls. Even rats receiving 3300 ppm failed to reach the weight of the untreated animals. On the other hand, the effects of the treatments on survival were not severe. At 6500 ppm, more than 85 % of male rats and female rats were alive at week 90, and 70 % were alive at termination of the study. Many neoplasms were found in the cyclohexanone-treated rats, but few of these differed in incidence from the untreated controls. Those that did, included neoplasms of the mammary glands and stromal polyps of the uterus in females, which were fewer in the treated groups than in the controls. The incidence of neoplasms of the liver, hepatocellular carcinomas and neoplastic nodules, did not differ significantly between cyclohexanone-treated rats and untreated controls. The incidence of these liver neoplasms was within the range of historical controls in the facility and in others. There were no non-neoplastic lesions in the rats that differed significantly in incidence between treated and untreated animals that could be attributed to the treatment with cyclohexanone. Based on the above results NOAEL of 462 mg/kg bw/d was determined.

 

Methyl methacrylate (2 -Generation reproduction study)

With regard to methyl methacrylate, twenty-five male and female Wistar rats (P parental generation) were administered with it at 0; 50; 150 and 400 mg/kg body weight/day per gavage in an OECD 416 2-generation reproductive toxicity study (BASF SE, 2009). At least 73 days after the beginning of treatment, P animals were mated to produce a litter (F1). Mating pairs were from the same dose group and F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were treated with the test substance at dosages of 0; 50; 150 and 400 mg/kg body weight/day post weaning, and the breeding program was repeated to produce F2 litter. No signs of systemic toxicity were observed other than slightly reduced body-weight gain at 150 mg/kg bw/d and above in the 2-gen study. Reduced body weights were judged to be the direct result of reduced food intake and therefore not an adverse finding. Consequently, the NOAEL was confirmed at the highest dose studied (400 mg/kg/d).

 

Methyl methacrylate (chronic repeated dose toxicity study)

Twenty-five male and female Wistar rats were administered three doses of methyl methacrylate in the drinking water for two years (Borzelleca et al. 1964). Initial doses of 6, 60 or 2000 ppm were partially raised to 7, 70 and 2000 ppm after 5 months.

A special design was employed to reduce the volatilization and measurements which showed that the methyl methacrylate concentrations remained within 15 % of the nominal concentration for 72 hours. Body weight depression was observed at 2000 ppm but it did not persist beyond the first few weeks of the study. Significant depression of fluid consumption was observed at 2000 ppm, although this tended to regress at the end of the study. Individual observations of depressed food consumption tended to parallel periods of depressed growth. There were significantly increased kidney weight ratios for female rats at 2000 ppm. These effects were believed to be a consequence of reduced food intake and reduced body weights, and in the absence of any histopathology, were considered as not biologically relevant. Therefore the NOAEL is considered to be >= 2000 ppm, corresponding to 90.3 mg/kg bw/day and 193.8 mg/kg bw/day, for males and females, respectively, on the basis of treatment specific fluid consumption rates and body weights.

 

In addition, further sub-chronic studies (OECD 408) and also combined repeated dose and reproductive/developmental toxicity screening studies (OECD 422) in rats with members of the lower methacrylates were performed indicating comparable NOAEL values. (Please refer to the read across justification)

 

Inhalation repeated dose toxicity

For the inhalation route no repeated dose toxicity studies of cyclohexyl methacrylate are available, therefore studies of the hydrolysis products cyclohexanol, methacrylic acid and the lower methacrylate ester methyl methacrylate were used to assess the potential of cyclohexyl methacrylate for toxicity via inhalation in a weight of evidence approach.

 

Cyclohexanol (sub-chronic repeated dose toxicity study)

A combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening test similar to OECD guideline 422 was performed (US EPA, 2010). 15 animals/sex/dose were exposed to cyclohexanol vapours at concentrations of 0, 50, 150 or 450/400 ppm (0; 0.21; 0.61; 1.64/1.84 mg/L). For males the exposure time was 6 hours/day, 5 days/week over a period of 16 weeks (10 weeks premating). 5 animals/sex/dose were allowed to recover for a period of 4 weeks. For the females exposure time was 6 hours/day, 5 days/week for 13 weeks.

Treatment-related effects observed were temporary and transient prostration at a concentration of 450/400 ppm. Further, a slight increase in mortality (3 males and 2 females) occurred within the premating period, when the animals were exposed to 450 ppm. Due to the mortality seen in the high dose group the 450 ppm level was lowered at the beginning of the mating period to 400 ppm. No systemic effects occurred at the 150 or 50 ppm levels.

The NOAEC for this repeated-dose toxicity study was 0.61 mg/L (150 ppm = 610 mg/m³)

 

Methacrylic acid (sub-chronic repeated dose toxicity study)

In an OECD 413 guideline study (sub-chronic inhalation toxicity: 90-day) 10 male and female Sprague Dawley rats per test group were whole body exposed to a vapour of the methacrylic acid on 6 hours per working day for 90 days (65 exposures) (MAA Task Force, BASF SE, 2008). The target concentrations were 20, 40, 100 and 350 ppm (corresponding to 70, 141, 352 and 1232 mg/m³). A concurrent control group was exposed to conditioned air. Methacrylic acid induced signs of general toxicity as indicated by decreased body weight, body weight gain, food consumption and transiently food efficiency in the high concentration male animals. At a concentration as high as 350 ppm, the local irritating effect was marginal, indicated by the hypertrophy/hyperplasia of the respiratory epithelium in the nasal cavity of two female animals. Substance-related changes of the sexual organs were not noted in any of the exposed animals, nor were there any changes of sperm mobility and sperm head counts. No indication of specific target organ toxicity was observed. Under the current test conditions, the no-observed adverse effect concentration (NOAEC) of this study is 352 mg/m³ for the male and female rats.

 

n-Butyl methacrylate (sub-acute repeated dose toxicity study)

In an OECD Guideline 412 Repeated Dose 28-day inhalation study with n-butyl methacrylate treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 952 and 1891 ppm (5626 and 11175 mg/m³) concentration groups throughout the study. There were no treatment-related effects on body weight or feed consumption, haematological measurements or clinical chemistry. At 1891 ppm (11175 mg/m³) relative kidney weights were increased but serum BUN values (resulting in increased BUN: creatinine ratio) were only slightly increased and there was no corresponding histopathology. The NOAEC for systemic effects was 952 ppm (5626 mg/m³).Local effects of inflammation of the olfactory region of the nasal cavity at 952 and 1891 ppm (5626 and 11175 mg/m³) were observed in both sexes. The NOAEC was determined to be 310 ppm (1832 mg/m³) (Hagan et al., 1993).

The saturated vapor concentration of cyclohexyl methacrylate of 1275 mg/m³ (183 ppm) is below the local and especially the systemic NOAEC of n-BMA.

 

Methyl methacrylate (sub-chronic and chronic repeated dose toxicity studies)

Solomon et al. (Rohm & Haas, 1991) administered methyl methacrylate by whole-body inhalation exposure to 5 groups (27 rats/group) of presumed pregnant rats (Crl: CDBR) at concentrations of ca. 0 (control), 0.4, 1.2, 4.8, 8.3 mg/L (corresponding to 99, 304, 1178, and 2028 ppm) for 6 h/day on days 6-15 of gestation (GD) in a developmental toxicity study according to OECD 414.

 

The US National Toxicology Program conducted a series of repeat-exposure inhalation toxicity studies of increasing duration with methyl methacrylate (MMA) in male and female rats and mice. Effects on the nasal respiratory tract were seen in studies in rats and mice in studies of 14 weeks (Rohm & Haas, Battelle Pacific Northwest Lab, 1980). In the 14 week study 10 male and female rats and mice were exposed to MMA concentrations 0, 500, 1000, 2000, 3000 and 5000 ppm (6 h/day, 5 days/wk for 97 d).

 

In a combined chronic toxicity/ carcinogenicity study, male and female Fischer 344 rats were exposed to methyl methacrylate monomer vapours at ca. 0, 0.10, 0.41 and 1.64 mg/L (0, 25, 100 and 400 ppm), 6 h/day, 5 days/wk for 24 months (Lomax et al., 1997). Parameters monitored throughout the study included clinical signs, individual body weights, haematology, clinical chemistry and urinalyses. 10 rats per sex per exposure group were killed after 13 and 52 weeks of exposure and all surviving rats were killed during week 104-106. Mortality and haematological, clinical chemistry and urinalyses parameters were not affected by methyl methacrylate exposure.

 

Weight of Evidence conclusion on inhalation

Local effects

The nasal cavity was identified as the target organ for chronic toxicity in male and female rats exposed to lower methacrylates as well as methacrylic acid. The microscopic nasal cavity changes occurred primarily in olfactory epithelium lining the dorsal meatus and consisted of degeneration of neuro-epithelium, basal cell hyperplasia and atrophy of Bowman's glands. In the inhalation studies local degeneration of the olfactory epithelia was observed in acute (6hrs) at 100 ppm, through to chronic (2-yr) studies at 100 ppm (LOEL) with marked degeneration at 400 ppm and above with methyl methacrylate and methacrylic acid. Histopathological changes with n-butyl methacrylate started to evolve at 952 ppm and were significantly distinct at 1891 ppm. No local effects were observed in the sub-chronic inhalation study of the hydrolysis product cyclohexanol.

Based on limited data from human tissue samples (that may not have been morphologically normal taken at polyp biopsy), the activity of alpha-naphthylbutyrate carboxylesterase in human nasal respiratory tissue is less than that in the rat (Mattes & Mattes, 1992). In addition, rodents are obligate nose breathers, whereas humans can also breathe through their mouth, which is expected to reduce exposure of the nasal epithelium. There are also differing nasal flow patterns, with the greater airflow across the human olfactory epithelium during the expiratory phase when the vapour concentration would be considerably reduced as a result of absorption in the lower respiratory tract. Furthermore, there are significant morphological differences between species in the structure of the nasal cavity, which result in differences in concentrations of inhaled materials at the nasal tissue.  These are reflected in differences in surface area normalized to minute ventilation, being fivefold greater in rodents than in humans (DeSesso, 1993). A much greater percentage of the nasal cavity is lined by olfactory epithelium in rats than in humans.

 

Effects on body weight

Slightly reduced body-weight gain was observed in the inhalation studies at 350 ppm and above. Methacrylates have a pungent odour (odour threshold 0.5 – 1.5 ppm) and can taint food and water. In the inhalation developmental toxicity study with methyl methacrylate reduced maternal body weight or decreases in maternal body-weight gain and decreases in maternal feed consumption were noted at 99 ppm and above in the absence of any other signs of general systemic toxicity up to the highest concentration tested (2028 ppm) (Solomon, 1991) clearly establishing that the reduced body weight gain was a consequence of reduced feed consumption.

From the histopathology and from clinical observations in the sub-chronic inhalation studies with n-butyl methacrylate, methacrylic acid and methyl methacrylate, it can be concluded that animals exposed to 350 ppm and above in the chronic inhalation study had substantial nasal mucosal irritation, increased nasal secretions and some discomfort and that this would have likely been expressed as a diminished sense of smell/behavioural changes. By analogy to the Solomon study it may be concluded that these changes would have the consequence of reducing appetite and have caused the decreased food consumption and depression of body-weight gain.

The association of olfactory epithelial injury and reduced feed consumption as well as decreased body-weight in high-level exposure groups has also been recognized in the literature for other chemicals.

For example, Dorman et al (2004) (Hirsch AR, and Zavala, 1999) reported that rats (F344 and Sprague-Dawley) and mice (B6C3F1) exposed to another olfactory epithelial toxicant, hydrogen sulphide (H2S), had exposure-related olfactory epithelial injury and rhinitis at exposure concentrations >= 30 ppm H2S. Rats exposed to 80 ppm ofH2S demonstrated reduced feed consumption and depressed terminal body weights.

In summary, the reduced body-weight gain observed in inhalation studies with methacrylic acid and the lower methacrylates is a direct consequence of reduced food intake, as a consequence of severe nasal irritation, reduced olfactory function and bad taste. These effects are not based on systemic toxicity. Hence, protecting against local effects (irritation and tissue damage in the respiratory tract) would protect against reduced body-weight gain in humans. No body weight effects were observed in the sub-chronic inhalation study with cyclohexanol.

 

Systemic effects

Signs of general systemic toxicity including histopathological changes in liver, kidneys, spleen, bone marrow and brain were seen at concentrations of 2000 ppm and above by inhalation.

After a 14-week inhalation of methyl methacrylate, rats showed splenic follicular atrophy and bone marrow atrophy at ca. 20.8 mg/L (5000 ppm; Battelle 1980). Malacia and gliosis of the brain in the 14 week range finder to the NTP study (Battelle, 1980) is considered being the relevant systemic effect, which was seen in 5/9 female rats exposed at 2000 ppm and 1/8 females at 1000 ppm. Therefore, the absence of this effect at 400 ppm (1640 mg/m³) in the 2-year study (Lomax et al., 1997) is considered representing the NOAEC for chronic systemic effects of methyl methacrylate.

For n-butyl methacrylate relative kidney weights were increased but serum BUN values (resulting in increased BUN: creatinine ratio) were only slightly increased and there was no corresponding histopathology at 1891 ppm (11175 mg/m³). The NOAEC for systemic effects was 952 ppm (5626 mg/m³).Local effects of inflammation of the olfactory region of the nasal cavity at 952 and 1891 ppm (5626 and 11175 mg/m³) were observed in both sexes. The NOAEC for local effects was determined to be 310 ppm (1832 mg/m³) (Hagan et al., 1993).

Both hydrolysis products did not show any dose related effects when inhaled. For cyclohexanol treatment-related effects observed were temporary and transient prostration at a concentration of 450/400 ppm. No effects occurred at the 150 or 50 ppm levels. The NOAEC for this repeated-dose toxicity study was set to 610 mg/m³ (150 ppm) (MPI Research Inc., 2005). For methacrylic acid a NOAEC of 352 mg/m³ (corresponding to 100 ppm) was determined based on histopathological changes due to local effects in the nasal cavity (goblet cell hypertrophy/hyperplasia in the respiratory epithelium) at higher doses.

 

Overall conclusion

Based on the observed effects with the read-across substances in the weight of evidence approach, the NOAEC for repeated inhalation of the test substance cyclohexyl methacrylate is expected to be in the same ranges. An oral repeated dose toxicity study with cyclohexyl methacrylate is available and the NOEAL of 300 mg/kg bw/d can be considered comparable to the values obtained in the read-across inhalation studies. Therefore the oral NOAEL is considered the most reliable value for cyclohexyl methacrylate and is used for inhalative risk assessment.

 

Other references

Dorman, Struve MF, Gross EA, and Brenneman KA. Respiratory tract toxicity of inhaled hydrogen sulfide in Fischer-344 rats, Sprague-Dawley rats, and B6C3F1 mice following subchronic (90-day) exposure. Toxicol Appl Pharmacol 198: 29-39, 2004.

 

Hirsch, and Zavala G. Long term effects on the olfactory system of exposure to hydrogen sulfide. Occup. Environ. Med. 56:284-287, 1999.

 

DeSesso JM. The relevance to humans of animal models for inhalation studies of cancer in the nose and upper airways. Qual Assur: Good Pract Regul Law 2: 213-231, 1993.

 

Mattes PM, Mattes WB. Naphthyl butyrate carboxylesterase activity in human and rat nasal tissue. Toxicol Appl Pharmacol, 114:71–76, 1994.

Justification for classification or non-classification

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

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.