2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

Endpoint summary

Description of key information

In Vivo Skin irritation: Not irritating to rabbit skin. 
In Vitro Eye Irritation: Not irritating in the BCOP assay. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 18 - April 2, 1993.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP and reported with a GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species: rabbit
Strain: White New Zealand
Source: Harald Schriever; Kaninchenfarm, D-2740 Bremervörde, Neuendamm 88
Animal selection: random
Animal identification: with individual ear tags; cage labelled with ear tag no., sex, date of study initiation, project no.

Husbandry
Housing: individual housing (50 x 45 x 40 cm, L x B x H) in a battery of cages, each equipped with a paper roll disposal system
Illumination: artificial lighting (120 lux) from 7.00 a.m. - 7.00 p.m.
Temperature: 20 ± 3° C
Relative humidity: 30 - 70 %
Measurement: with thermohygrometer twice daily

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5g

Duration of treatment / exposure:

4 hour

Observation period:

72 hours

Number of animals:

3 animals

Details on study design:

Preparation if the animals
24 h before treatment, fur was removed with electric clippers from an area of roughly 8 x 15 cm on the back of each animal. The skin was examined for abrasions and only animals with healthy, intact skin were used in the test.

Preparation of the test article
The test article was moistened sufficiently with Aqua ad iniectabilia to ensure a good contact with the skin.

Exposure
In each animal, 0.5 g of the test article were applied to the test site (ca. 6 cm2 in size), an adjacent area of untreated skin serving as a control. Each test area was covered with a semi-occlusive dressing consisting of Ypsiplast® (Holthaus Medical, Remscheid-Lüttringhausen), which was held in place by non-irritating tape Elastoplast® (Beiersdorf AG, Hamburg), and Stülpa® (P. Harmann AG, Heidenheim-Brenz), which enveloped the whole of the animal's trunk. At the end of the 4-h exposure period, the dressing was removed and any residual sample was carefully washed off with water or an appropriate solvent.

Observations
Animals were examined for signs of skin reactions at 30-60 min, 24, 48 and 72 h after patch removal. The skin was evaluated according to the scheme presented in table form (see Any other information).

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 0, 24, 48 & 72h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 0, 24, 48 & 72h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No test artcle-dependent findings were observed.

Other effects:

No toxic effects were observed.

Individual values of skin reactions

Animal number	Time after patch removal
	30-60 min		24h		48h		72h
	Ery	Oed	Ery	Oed	Ery	Oed	Ery	Oed
	T C	T C	T C	T C	T C	T C	T C	T C
1	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
2	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
3	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0

T = test site

C = control site

Ery = erythema

Oed = oedema

 

Mean values of skin reactions of 24, 48 and 72h after patch removal

Animal number	Erythema (test site)	Oedema (test site)
1	0.0	0.0
2	0.0	0.0
3	0.0	0.0

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The mean grades of skin reactions at 24, 48 and 72 h after patch removal were lower than the value classified as irritant by the EEC directive 91/325/EEC of March 1, 1991. When applied to the skin, the test article "Lowinox PSW" may therefore be considered to be non-irritant.

Executive summary:

The aim of the test was to assess the irritant and/or corrosive effect(s) of "Lowinox PSW" on the skin. Information derived from this test serves to indicate the existence of possible hazards likely to arise from exposure of the skin to the test article.

The test was conducted according to the OECD guideline for the testing of chemicals no. 404 (May 12, 1981) and the EEC directive 84/449/EEC (September 19, 1984). The study was carried out as described in the corresponding protocol approved by the testing facility and the study sponsor on February 12 and 24, 1993, respectively. The principles of Good Laboratory Practice for the testing of chemicals as specified by national (BGB1. I, no. 13, § 19a, March 22, 1990) and international (OECD, Paris, 1982) legislation were followed during the performance of the study.

 

SUMMARY

The potential toxicity of "Lowinox PSW" was assessed in an acute dermal irritation/corrosion test on 3 albino rabbits. The skin was exposed to the test article for 4 h. Animals were examined for signs of erythema and oedema at 30-60 min, 24, 48 and 72 h after patch removal.

 

Results

No test article-dependent findings were observed.

 

The mean grades of skin reactions at 24, 48 and 72 h after patch removal were lower than the value classified as irritant by the EEC directive 91/325/EEC of March 1, 1991. When applied to the skin, the test article "Lowinox PSW" may therefore be considered to be non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2015 to 13 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

not specified

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter, but within maximum 4 hours.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

318.0 to 333.7 mg

Duration of treatment / exposure:

240 minutes of treatment.

Observation period (in vivo):

No observation period required for this study.

Number of animals or in vitro replicates:

3 eyes per group.

Details on study design:

Reference substances
Negative control: A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Study design
Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas, 3 corneas per treatment group, were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (range for 3 eyes 318.0 to 333.7 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg
Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Irritation parameter:

cornea opacity score

Run / experiment:

After 240 minutes treatment

Value:

-0.3 - 7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

After 240 minutes of treatment

Value:

-0.4 - 1.2

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints.

Other effects:

None specified

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1,2
Negative control	0.0	0.000	0.0
Positive control	84.3	3.975	144.0
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]	0.0	0.019	0.3

¹Calculated using the negative control mean opacity and mean permeability values

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value)

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

Opacity score

Eye	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity^2	Mean Opacity
Negative control
16	1	1	0	-0.3	0.0
17	1	2	1	0.7
18	2	2	0	-0.3
Mean final opacity: 0.3
Positive control
1	0	80	80	79.7	84.3
2	0	74	74	73.7
3	1	101	100	99.7
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
13	2	2	0	-0.3	0.0
14	1	2	1	0.7
15	1	1	0	-0.3

¹Final Opacity = Opacity after treatment – Opacity before treatment

²Negative control correct Final Opacity = Final opacity – Mean final opacity negative control

 

Permeability score individual values (uncorrected)

Eye	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control
16	1	0.012	0.012	0.013	0.012	0.012	0.011
17	1	0.011	0.002	0.003	0.005	0.005
18	1	0.014	0.017	0.015	0.015	0.015
Positive control
1	6	0.833	0.831	0.832	0.832	4.992
2	6	0.528	0.532	0.546	0.535	3.212
3	6	0.674	0.649	0.637	0.653	3.920
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
13	1	0.009	0.009	0.009	0.009	0.009
14	1	0.047	0.046	0.050	0.048	0.048
15	1	0.027	0.028	0.043	0.033	0.033

 

Permeability score individual values (corrected)

Eye	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OC490Average	Negative control corrected final OD490	Average OD
Negative control
16	1	0.001	0.001	0.002	0.001	0.001	0.000
17	1	0.000	-0.009	-0.008	-0.006	-0.006
18	1	0.003	0.006	0.004	0.004	0.004
Positive control
1	6	0.822	0.820	0.821	0.821	4.926	3.975
2	6	0.517	0.521	0.535	0.524	3.146
3	6	0.663	0.638	0.626	0.642	3.854
2,2’-methylenebis[6-(1-methylcycloheyl)-p-cresol]
13	1	-0.002	-0.002	-0.002	-0.002	-0.002	0.019
14	1	0.036	0.035	0.039	0.037	0.037
15	1	0.016	0.017	0.032	0.022	0.022

¹OD₄₉₀values corrected for the mean final negative control permeability (0.011).

 

In Vitro irritancy score

Eye	Negative control corrected Final Opacity	Negative control corrected Final OD490	In vitro Irritancy Score^1
Negative control
16	-0.3	0.001	-0.3
17	0.7	-0.06	0.6
18	-0.3	0.004	-0.3
Positive control
1	79.7	4.926	153.6
2	73.7	3.146	120.9
3	99.7	3.854	157.5
2,2’-methylbis[6-(1-methylcyclohexyl)-p-cresol]
13	-0.3	-0.002	-0.4
14	0.7	0.037	1.2
15	-0.3	0.022	0.0

¹In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

 

Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-3 – 2	-0.056 – 0.050	-3.3 – 2.1	68 – 135
Mean	-0.22	0.00	-0.28	95.22
SD	0.94	0.01	0.95	18.75
N	94	96	93	3

SD = Standard deviation

N = Number of observations

The above mentioned historical control data range of the controls were obtained by collected all data over the period April 2012 to April 2015.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.3 after 240 minutes of treatment.

Executive summary:

Evaluation of the eye hazard potential of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

This report evaluates the potency of chemicals to induce serious eye damage using isolated bovine corneas. The potential eye damaging effects of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was tested through topical application for approximately 240 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 437: " Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”(adopted July 26, 2013).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.

 

Batch WWP4J0001 of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was a white to cream powder. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas of three eyes (range for 3 eyes 318.0 to 333.7 mg).

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 144 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.3 after 240 minutes of treatment.

Since 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vivo Skin Irritation:

The aim of the test was to assess the irritant and/or corrosive effect(s) of "Lowinox PSW" on the skin. Information derived from this test serves to indicate the existence of possible hazards likely to arise from exposure of the skin to the test article.

 

SUMMARY

The potential toxicity of "Lowinox PSW" was assessed in an acute dermal irritation/corrosion test on 3 albino rabbits. The skin was exposed to the test article for 4 h. Animals were examined for signs of erythema and oedema at 30-60 min, 24, 48 and 72 h after patch removal.

 

Results

No test article-dependent findings were observed.

 

The mean grades of skin reactions at 24, 48 and 72 h after patch removal were lower than the value classified as irritant by the EEC directive 91/325/EEC of March 1, 1991. When applied to the skin, the test article "Lowinox PSW" may therefore be considered to be non-irritant.

In Vitro Eye Irritation:

The Bovine Corneal Opacity and Permeability Assay (BCOP) is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.

 

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

 

Evaluation of the eye hazard potential of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

This report evaluates the potency of chemicals to induce serious eye damage using isolated bovine corneas. The potential eye damaging effects of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was tested through topical application for approximately 240 minutes.

 

Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas of three eyes (range for 3 eyes 318.0 to 333.7 mg).

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 144 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.3 after 240 minutes of treatment.

Justification for selection of skin irritation / corrosion endpoint:
Study performed in a GLP accredited laboratory in accordance with OECD Guideline 404 and EU Method B.4.

Justification for selection of eye irritation endpoint:
Study performed in a GLP accredited laboratory in accordance with OECD Guideline 437 and EU Method B.47.

Justification for classification or non-classification

In Vivo Skin Irritation:

The mean grades of skin reactions at 24, 48 and 72 h after patch removal were lower than the value classified as irritant by the EEC directive 91/325/EEC of March 1, 1991. When applied to the skin, the test article "Lowinox PSW" may therefore be considered to be non-irritant.

In Vitro Eye Irritation:

Since 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage