Registration Dossier

Administrative data

Description of key information

LD50 = 2500 mg/kg bw | rat (male/female) | OECD 423 | 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Principles of method if other than guideline:
DEVIATION
The starting dose was selected to be 300 mg/kg body weight. No compound-related mortality was recorded for all animals of step 1. As only slight signs of toxicity were noted a further step at this dose level was omitted for animal welfare reason - however opposed to OECD Guideline 423 (Annex 2c).
Instead, the second step was performed at a dose of 2000 mg/kg body weight. No compound-related mortality was recorded for any animal of step 2.
Based on these results and according to the acute toxic class method regime, a third step was performed with animals at a dose of 2000 mg/kg body weight. As opposed to previous steps where female animals were used, confirmatory step 3 was performed with male animals.
Compound-related mortality was recorded for one animal of step 3. Based on these results and according to the acute toxic class method regime no further testing was required.

REASON
Further testing of 3 animals at 300 mg/kg body weight was omitted for animal welfare reason. As confirmed by the results at 2000 mg/kg body weight, these animals would not have had an effect on classification of the test item.
The use of male animals in confirmatory step no. 3 was chosen on the assumption that a genderspecific susceptibility might be obtained.
This deviation did not influence the quality or integrity of the present study.

GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS

- Species/strain: WISTAR rats Crl: WI(Han)
- Source: Charles River
- Sex: Step 1-2 female (non-pregnant and nulliparous), Step 3 male
- Number of animals: 3 per step
- Age at the beginning of the study: 8-12 weeks old
- Body weight on the day of administration:
Step 1: animals no.: 1 - 3: 150 - 159 g (females)
Step 2: animals no.: 4 - 6: 166 - 172 g (females)
Step 3: animals no.: 7 - 9: 200 - 256 g (males)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

The animals were marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test itemwas administered. Food was provided again approximately 4 hours post dosing.

HOUSING & FEEDING

- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 per hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1526)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 131113)
- Certificates of food, water and bedding are filed at the test institute
- Adequate acclimatisation period (at least five days) under laboratory conditions
Route of administration:
oral: gavage
Vehicle:
corn oil
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
300 mg/kg: 3 females
2000 mg/kg: 3 females + 3 males
Control animals:
no
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
2 500 mg/kg bw
Mortality:
yes, 1 male (200 mg/kg bw dosing) euthanized for ethical reasons
Clinical signs:
The following clinical signs have been observed throughout the study:
Spontaneous activity reduced (slightly to moderately), Bradykinesia, Piloerection (slight to moderate), Eyes closed or half closed, Salivation (slight to moderate), Wasp waist, Kyphosis, Moving the bedding
Body weight:
None of the animals showed weight loss during the observation period.
Mean bodyweight gain on day 15 (compared to day 1): Step 1 18.7 %; Step 2 15.0 %; Step 3 20.5 %.
Gross pathology:
With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

CLINICAL SIGNS PER STEP

STEP 1: 300 mg/kg bw, 3 females

 0 minutes  nsf
 60 minutes  Spontaneous activity slightly reduced, Piloerection (slight)
 2 d - 15 d  nsf

STEP 2: 2000 mg/kg bw, 3 females

 0 minutes  nsf
 60 minutes  Spontaneous activity moderately reduced, Kyphosis, Piloerection (moderate), Eyes closed, Wasp waist
 2 d - 15 d  nsf

STEP 3: 2000 mg/kg bw, 3 males

 0 minutes  nsf
 60 minutes  Spontaneous activity moderately reduced, Piloerection (moderate), Eyes closed, Wasp waist, Bradykinesia, Salivation moderate, Moving the bedding
 2 d - 15 d  2 males nsf, 1 male euthanized for ethical reasons

LD50 CUT-OFF

 Dose [mg/kg bw]  No. of animals  No. of intercurrent deaths  LD50 Cut-Off
 2000  6  1   2500 mg/kg bw
 300  3  0
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, a single oral application of the test item 3,4-Epoxycyclohexylmethyl methacrylate to female rats at a dose of 300 mg/kg body weight was associated with slight signs of toxicity but no mortality.
Under the conditions of the present study, a single oral application of the test item 3,4-Epoxycyclohexylmethyl methacrylate to rats at a dose of 2000 mg/kg body weight was associated with signs of toxicity and mortality, which were slight more prominently expressed in male compared to female animals.

The median lethal dose of 3,4-Epoxycyclohexylmethyl methacrylate after a single oral administration to female and male rats, observed over a period of 14 days is: LD50 cut-off (rat): 2500 mg/kg bw

In conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC [7] the test item has no obligatory labelling requirement for toxicity.
According to Annex I of Regulation (EC) 1272/2008 [8] the test item 3,4-Epoxycyclohexylmethyl methacrylate has no obligatory labelling requirement for toxicity and is not classified.
According to GHS (Globally Harmonized Classification System) [9] the test item 3,4-Epoxycyclohexylmethyl methacrylate has obligatory labelling requirement for toxicity and is classified into Category 5.
Executive summary:

SUMMARY

One group, with three female WISTAR Crl: WI(Han) rats, was treated with the test item by oral gavage administration at a dosage of 300 mg/kg body weight. The test item was suspended with the vehicle corn oil at a concentration of 0.03 g/mL and administered at a dose volume of 10 mL/kg.

Two groups, one female and one male group each of three WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended with vehicle corn oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at the test institute were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

 Step

 Sex / No.

 Starting dose [mg/kg]

No. of animals

 No. of intercurrent deaths

 1

 female / 1 -3

 300

 3

 0

 2

 female / 4 -6

 2000

 3

 0

 3

 male / 7 -9

 2000

 3

 1

All female animals treated with the test item at a dose of 300 mg/kg and 2000 mg/kg survived until the end of the study showing test-item related signs of toxicity.

One male animal treated with the test item at a dose of 2000 mg/kg had to be sacrificed for ethical reasons on test day 1. All remaining male animals survived until the end of the study.

The most relevant clinical findings in the animals treated with the test item at a dose of 300 mg/kg bw were reduced spontaneous activity and piloerection. All symptoms recovered within the day of treatment.

The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, bradykinesia, piloerection, eyes closed, eyes half closed, salivation, moving the bedding and kyphosis. All symptoms recovered within up to the 2nd day post-dose. The clinical symptoms were similar in both genders, however, slightly more prominently expressed in male compared to female animals.

Throughout the 14-day observation period, the body weight gain of the surviving animals was within the normal range of variation for this strain.

Macroscopic findings of surviving animals: At necropsy, no treatment-related macroscopic findings were observed in any animal of any step. Macroscopic findings of animals not having survived until the end of the observation period: At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
2 500 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute oral toxicity:

3,4-Epoxycyclohexylmethyl methacrylate was tested for acute oral toxicity in a GLP-compliant study according to OECD TG 423 (Klimisch 1). Under the conditions of the study, a LD50 of 2500 mg/kg body weight was derived.

Acute inhalation toxicity:

An acute inhalation study is not available for 3,4-epoxycyclohexylmethyl methacrylate and was omitted for the following considerations:

The exposure via inhalation is limited by the physico-chemical properties of the substance, such as the rather low vapor pressure (<19 mbar or <1.9 kPa) at 20°C) and a high boiling point (>117°C). Furthermore, the expected exposure scenarios do not imply spraying applications in industrial / professional / consumer settings. The substance is handled under controlled conditions at industrial workplaces, where inhalation exposure to very small quantities of vapors or aerosols could theoretically arise during the transfer of the substance into reactors. However, in consideration of the mutagenic properties of 3,4-epoxycyclohexylmethyl methacrylate, adequate risk management measures (RMM) are already implemented to prevent exposure by any route. These RMM include local exhaust ventilation as well as the use of protective gloves, protective goggles and respiratory masks. In conclusion, inhalation is not a relevant route of exposure during the intended use of 3,4-epoxycyclohexylmethyl methacrylate.

Acute dermal toxicity:

An acute dermal study is not available for 3,4-epoxycyclohexylmethyl methacrylate and was omitted for the following considerations:

An acute oral toxicity test (OECD TG 423, GLP) was conducted with 3,4-epoxycyclohexylmethyl methacrylate. On the basis of this test, the substance does not meet the criteria for classification as acute oral toxicant. Signs of systemic toxicity observed in the study indicate that oral absorption took place. The low molecular weight (appr. 200 Da), the log Pow (2.4) and the high water solubility (3.35 g/L) are further indicators for a good absorption via the gastrointestinal tract. The same PC parameters favor also the dermal penetration, though in contrast to the gastrointestinal tract, the skin represents an additional barrier. 3,4-Epoxycyclohexylmethyl methacrylate was shown to be a skin irritant in vitro, but no signs of skin irritation were detected in the in vivo skin sensitization assay (LLNA) in mice. A significant contribution to skin permeability due to the induction of skin damage is therefore unlikely. Expecting a similar or rather lower systemic bioavailability after dermal compared to oral uptake, the acute oral toxicity test of 3,4-epoxycyclohexylmethyl methacrylate can serve as a basis to conclude that the substance has not to be classified as acute dermal toxicant.

Further evidence for the lack of an acute dermal toxic potential is provided by the skin sensitization study (LLNA) in mice. In this test, the animals were treated with topic applications of each up to 2.6 g/kg body weight/day on three consecutive days. The applied doses induced no mortality but clinical symptoms, which indicate that 3,4-epoxycyclohexylmethyl methacrylate was systemically bioavailable after dermal application. On the basis of the LLNA study in mice, a dermal LD50 of >2000 mg/kg could be expected for rodents.

Finally, dermal exposure during the use of 3,4-epoxycyclohexylmethyl methacrylate is unlikely. As the substance is considered to be a skin sensitizer and a mutagen, dermal contact is prevented by the use of protective gloves at the industrial workplace.

Justification for classification or non-classification

The median lethal dose (LD50) was found to be above 2000 mg/kg bw after oral application of 3,4-epoxycyclohexylmethyl methacrylate to rats. Consequently, a classification under the EU regulation (Regulation (EC) 1272/2008) is not required. The substance has to be classified in category 5 for acute toxicity under the Globally Harmonized System of Classification and Labelling of Chemical Substances of the United Nations (UN-GHS).

With regard to inhalation or dermal exposure, classification as acute toxic seems not to be justified on the basis of an expert judgement considering the result of all available studies, exposure scenarios and physico-chemical properties.