Registration Dossier

Administrative data

Description of key information

2,4-Diaminomethylcyclohexane shows a corrosive potential in the EpiDerm™ skin corrosivity test (BASF SE, 2009; Val 1) and causes skin and cornea necrosis in rabbits (Smyth et al., 1969).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-06-17 - 2011-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)
Qualifier:
according to guideline
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
adopted July 19, 2006
Deviations:
no
GLP compliance:
yes
Species:
other: in vitro test on a reconstituted collagen matrix (Corrositex® Biobarrier Membrane).
Strain:
other: not applicable (in vitro test)
Details on test animals or test system and environmental conditions:
Not applicable
Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 500 μL of the test substance was added onto the membrane disc
Duration of treatment / exposure:
Not applicable
Observation period:
until breakthrough of the test substance through the membrane disc coated with the biobarrier matrix.
Number of animals:
not applicable
Details on study design:
I TEST SYSTEM
- The Corrositex® assay kit is commercially available from InVitro International.
- The assay is based on the time that is required for the test substance to penetrate through the Corrositex® Biobarrier Membrane and produce a change in the Chemical Detection System (CDS).
- The Corrositex® assay is used to determine the corrosive potential of test substances. The assay is limited to testing those materials which cause detectable pH changes in the CDS
II Pretext
- Qualification screen (to determine if a color change can be detected): 150 μL of the test substance was added to the CDS screening tube. If the test substance failed to produce a color change in the CDS within one minute, the test substance could not be analyzed in this system, and no further testing was required.
- Categorization screen (to categorize weak acids/bases and strong acids/bases): the categorization screen was performed by adding 150 μL of test substance to each tube A and B. Each tube was mixed and the resulting color observed. If required, 2 drops of the "confirm" reagent were added to tube B, the tube mixed, and the resulting color observed. The test substance was scored as category 1 (high acid/alkaline reserve) or category 2 (low acid/alkaline reserve).

III MAIN TEST (Corrositex®)
- Biobarrier preparation: the vial containing the biobarrier matrix powder was placed in a water bath at 64 – 68ºC. The entire content of the biobarrier diluent vial was added slowly to the matrix powder (stir bar rotation) to avoid foaming of the solution. 200 μL of the solubilized matrix was pipetted into each of the membrane discs, and the membrane discs were then refrigerated for at least 2 hours at 2 – 8ºC. The biobarriers were wrapped and stored at 2 – 8ºC for a maximum of 7 days.
- Corrositex® assay (after acceptance of the positive control)
Four vials containing the CDS were used for the test substance. In addition, one vial was used for the PC, NC and for the color (blank) control, each.
a) A membrane disc coated with the biobarrier matrix was placed into one vial containing the CDS and approximately 500 μL of the test substance was added onto the membrane disc. An electronic time clock was started with the application. The vial was observed for three minutes for any change in the CDS.
b) If no color change was observed within three minutes, the remaining membranes were treated with the test substance. An electronic time clock was started with each application. The vials were observed continuously for the first ten minutes. Thereafter the vials were observed for approximately ten minutes around the time points relevant for evaluation or until breakthrough of the test substance occurred. The elapsed time between test-substance application and the first change in the indicator solution (i.e. barrier penetration) was recorded.
c) The positive control vial was prepared as described above and received one pellet of sodium hydroxide on top of the membrane disc. This vial was monitored continuously until breakthrough had occurred.
The negative control vial was prepared as described above and received 500 μL of 10% citric acid. This vial was observed for 60 minutes and was evaluated as “non-corrosive” if no reaction had been observed.

IV SCORING SYSTEM
- Corrosive potential was determined on the basis of the average time recorded for the test substance to produce a change in the CDS (see Table 1).
- Acceptance criteria: The Corrositex® assay was accepted if the breakthrough time for the positive control substance was in the historic control range (mean ± 2-3 x standard deviations). To demonstrate the functional integrity of the membrane barrier, the acceptance criterion for the negative control was not to induce membrane breakthrough within a 60 min observation period.
Irritation / corrosion parameter:
penetration time (in minutes)
Value:
6.3

Table 2: Summary of the findings

 

Breakthrough time (min:s)

 

Vial 1

Vial 2

Vial 3

Vial 4

Mean

Test substance

5:01

4:35

7:30

8:10

6:19

Positive control

10:14

-

-

-

-

Negative control

NB

-

-

-

-

NB = no break through within maximum observation period (60 min)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study meets generally accepted scientific principles (restriction: purity unknown, limited documentation). The registered substance is a reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen (common name: diaminomethylcyclohexane or MDACH, respectively) consisting of the two isomers 4 -methylcyclohexane-1,3-diamine (CAS 13897-55-7; molecular weight approx. 128 g/mol) and 2-methylcyclohexane-1,3-diamine (CAS 13897-56-8; molecular weight approx. 128 g/mol). The registered substance was tested in several toxicity studies, which were conducted on the one hand using 4-methylcyclohexane-1,3-diamine (CAS 13897-55-7) which is the main isomer of the registered substance (up to 90%) and on the other hand the reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen was tested as such (for details see read across justification, IUCLID chapter 13).
Qualifier:
no guideline followed
Principles of method if other than guideline:
To aevaluate the eye irritation potency of the test substance, a flooding volume (0.5 ml) was instilled directly into the eye of 5 rabbits (not rinsed). Eye injury in rabbits was then recorded in a 10-grade ordinal series and is based upon the degree of corneal necrosis that results from instillation of various volumes and concentrations of chemical.
American J. Ophthalmology 29: 1363.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
not reported


ENVIRONMENTAL CONDITIONS
not reported
Vehicle:
unchanged (no vehicle)
Remarks:
Where dilution of a chemical is necessary, the preferred solvent was propylene glycol from a batch shown to cause no injury, followed by water, and in some cases a deodorized kerosene known as "Deobase" has been used.
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.005, 0.02, 0.1 or 0.5 mL. Guided by the result and the table of injury grades below, additional applications are made until the chemical can be assigned to one of the defined grades. If large volumes are applied, the lids are held closed for one minute before the animal is released.
- Concentration (if solution): 1, 5, 15, 40 or 100%
Duration of treatment / exposure:
unrinsed
Observation period (in vivo):
18 - 24 h
Number of animals or in vitro replicates:
5
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
The individual numerical scores of eye treated with a given volume or concentration of a chemical are added together and then divided by the number of eyes (usually 5) to obtain the score injury caused by the treatment. The level of 5.0 was selected as representative of severe injury. This figure corresponds to necrosis visible only after staining and covering about three fourths of the surface of the cornea; or a more severe necrosis covering a smaller area.

1) Generally the symptoms were scored as followed:
- Symptom visible before Fluorescein staining:
cornea dull: 2 points
cornea opaque, less than half of area: 4 points
cornea opaque, more than half of area: 6 points (Maximum points)
Keratoconus: 6 points (Maximum points)
Iritis, slight internal congestion: 1 point
Iritis, marked internal congestion: 2 points (Maximum points)

- Symptom visible after Fluorescein staining:
Necrosis on less than 5% of cornea: 1 point
Necrosis on 5 to 12%: 2 points
Necrosis on 13 to 37%: 3 points
Necrosis on 38 to 62%: 4 points
Necrosis on 63 to 87%: 5 points
Necrosis on 88 to 100%: 3 points (Maximum points)

- Total maximum points: 20 points

2) The injury was then graded according to the following table:
- Grade 1: 0.5 ml undiluted gives injury of 0 to 1.0 points
- Grade 2: 0.5 ml undiluted gives injury of over 1.0 up to 5.0 points
- Grade 3: 0.1 ml undiluted gives injury of up to 5.0 points (0.5 ml gives over 5.0)
- Grade 4: 0.02 ml undiluted gives injury of up to 5.0 points (0.1 ml gives over 5.0)
- Grade 5: 0.005 ml undiluted gives injury of up to 5.0 points (0.02 ml gives over 5.0)
- Grade 6: excess of 40% solution gives injury of up to 5.0 points (0.005 ml gives over 5.0)
- Grade 7: excess of 15% solution gives injury of up to 5.0 points (40% gives over 5.0)
- Grade 8: excess of 5% solution gives injury of up to 5.0 points (15% gives over 5.0)
- Grade 9: excess of 1% solution gives injury of up to 5.0 points (5% gives over 5.0)
- Grade 10: excess of 1% solution gives injury of over 5.0 points

TOOL USED TO ASSESS SCORE: the eye is examined in strong diffuse daylight, then stained with fluorescein and the injury scored
Irritation parameter:
overall irritation score
Remarks:
irritation
Basis:
mean
Time point:
other: 18-24 hours after treatment
Score:
9
Max. score:
10
Reversibility:
other: not applicable (necrosis)
Remarks on result:
other: The test substance was attributed to Grade 9 on a scale of 10, indicating that excess of 1% solution gives injury of up to 5.0 points (5% gives over 5.0), corresponding to necrosis on 63 to 87% of the cornea.
Interpretation of results:
corrosive
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

1) In vitro studies

The potential of 2,4-Diaminomethylcyclohexane to cause dermal corrosion was assessed by a single topical application of 50 μL of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™; BASF SE, 2009, Val 1). Two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as suitable endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability.

 The EpiDerm skin corrosivity test showed the following results:

- Viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 26%.

- Viability of the test-substance treated tissues determined after an exposure period of 1 hour was 10%.

- The test substance was also able to directly reduce MTT. However, the induced direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

To further specify its degree of corrosivity for the purpose of classification, the test substance was also assessed for its potential to cause dermal corrosion by a single topical application of 500 μL of the test substance to the Corrositex® Biobarrier Membrane (Corrositex® assay). The Corrositex® Biobarrier Membrane is a test system consisting of a reconstituted collagen matrix. The assay is based on the time that is required for the test substance to penetrate through the Corrositex® Biobarrier Membrane and produce a change in the Chemical Detection System (CDS). In addition to the test substance a positive and a negative control were assessed.

The Corrositex® assay showed the following results: (1) the qualification screen demonstrated that the test substance is able to react with the CDS and produce a visible color change. Therefore the membrane barrier test method was determined to be suitable for the evaluation of the corrosive potential of the test substance. (2) A timescale category test was carried out to distinguish between weak and strong acids or bases. The test substance was assigned to timescale category 1 (having a high acid/alkaline reserve). (3) In the main test 4 Corrositex® Biobarrier Membranes were treated with the test substance. The mean breakthrough time of the test substance, determined in the actual Corrositex® assay, was 6 minutes and 19 seconds.

Based on the observed results and applying the evaluation criteria (time required for CDS change:  > 3 min, ≤ 1 hour), it was concluded that the test substance shows a corrosive potential in the Corrositex® - Skin Corrosion Test under the test conditions chosen. Based on the mean breakthrough time, the test substance has to be classified as corrosive (Cat 1B under UN GHS / CLP regulations, and corresponds to R34 according to according to 67/548/EEC).

 

2) In vivo studies

Only one poorly documented in vivo study is available, in which the primary skin irritation was evaluated in a 10-grade ordinal series for the severest reaction that develops on the clipped skin of each of five animals within 24 hours of the uncovered application of test substance (0.01 mL; undiluted or as solution in water, propylene glycol, or acetone) to the albino rabbit belly (Smyth et al., 1969; Val 2). The tested substance was attributed a grade score of 6, indicating that it causes necrosis when applied undiluted (no further details provided).

 

Eye Irritation

The eye irritation potency of 2,4-Diaminomethylcyclohexane was also evaluated in the Smyth publication (Smyth et al., 1969; Val 2). When a flooding volume (0.005, 0.02, 0.1 or 0.5 mL) of test substance at concentration levels of 1, 5, 15, 40 or 100% in propylene glycol, water or deodorized kerosene (not further specified) was instilled into the rabbit eye (5 animals, no washing) and observed 18-24 hours thereafter, the test substance was attributed to Grade 9 on a scale of 10, indicating that excess of 1% solution caused injury of up to 5.0 points (5% caused over 5.0), corresponding to necrosis on 63 to 87% of the cornea.

Respiratory irritation

No data; the test substance shows corrosivity potential in vitro and in vivo.


Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

2,4-Diaminomethylcyclohexane shows a corrosive potential in the EpiDerm™ skin corrosivity test under the test conditions chosen (BASF SE, 2009). Unfortunately, the test method does not yet allow for the differentiation of severity of the effect. The corrosivity potential of the substance was also confirmed in vivo, as evidenced by skin and cornea necrosis in rabbits after local treatment (Smyth et al., 1969). Further evidences obtained from the in vitro Corrositex® - Skin Corrosion Test (BASF SE, 2011) confirmed that the registered substance has to be classified as corrosive.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for skin and eye irritation (cat. 1B, H314) under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).