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Description of key information

Based on overall evidence from in vitro and in vivo studies, ammonium carbamate is not considered to be irritating to skin. Based on the available in vitro and in vivo studies, the substance is considered to cause serious damage to eyes. 

Key value for chemical safety assessment

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin Irritation

 

One GLP-compliant in vitro EpiDerm Skin Corrosion / Irritation Test, performed according to OECD guideline 431, was available for assessment (BASF SE, 2010a). Normal, human-derived epidermal keratinocytes, which have been cultured to form a multi layered, highly differentiated model of the human epidermis, were used. The corrosive potential of ammonium carbamate was predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with highly deionised water. A chemical is considered "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%. Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered an "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

In the corrosion test, two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator at 37 ˚C) and test group (test material, negative control and positive control; 12 tissues per test) were used. 25 μl highly de-ionised water was applied first. Thereafter, a volume of 25 μl of the test material was applied and homogeneously distributed with water. Control tissues were concurrently treated with 50 μl of highly de-ionised water (negative control) or with 50 μl of 8 N potassium hydroxide (positive control). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment, followed by 3-hour incubation with MTT solution.

In the irritation test, three tissues were treated with the test substance, the positive and negative controls, respectively. The test material was applied in the same way as in the corrosion test. Control tissues were concurrently treated with 30 μl of sterile PBS (negative control) or with 30 μl of 5% SDS (positive control). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator at 37 ˚C and washed with sterile PBS to remove residual test material afterwards. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours, followed by 18 ± 2 hours post-incubation period and subsequent 3 hours incubation with MTT.

In the corrosion test, the mean tissue viability was 98% of the negative control value after 3 minutes exposure and 18% of the negative control value after 1 hour exposure. For the positive control in the corrosion test, the mean tissue viabilities were 22 and 13% after 3 minutes and 1 hour exposure, respectively.

The mean tissue viability was 8% (SD = 0.47) of negative control in the irritation test (1 hour exposure duration). The mean tissue viability after 1 hour was 7% for the positive control. Based on these results, ammonium carbamate was considered to be irritating, but not corrosive to skin.

 

In addition, in a dermal irritation study (BASF, 1958), two Vienna White rabbits were treated with the test substance for 1, 5 and 15 minutes under occlusive conditions. The application sites were covered with either 40 or 80% preparations of ammonium carbamate (no data on purity) in water. Before skin changes were scored, the application sites were washed with aqueous solution of Lutrol 9. Very slight erythema was seen at all incubation time points upon removal of test patch. The effect disappeared within 24 hours. Since no skin irritation nor clinical symptoms were observed in any of the treated animals at this point, the study was terminated 24 hours after removal of the patches. Under the test conditions, the test substance did not give indications of an irritant property to the skin (BASF AG 1958). This study is well documented and meets general scientific standards, however it not adequate for the assessment of irritation as the maximum exposure duration of 15 min is too short in comparison to the OECD guideline 404 recommended exposure durations of 4 hours. Moreover, only dilutions and not the undiluted test substance were tested.

Finally, the irritative potential of the ammonium carbamate following a single, semi-occluded exposure to the skin of albino rabbits was determined in a GLP-compliant in vivo study conducted according to OECD guideline 404.

Initially, a single animal was dosed to evaluate the irritative potential of the test substance. This single rabbit received 3 applications of the test substance applied concurrently for 3 minutes, 1 hour, and 4 hours. Each 0.5-g dose of the test substance was moistened with deionized water and applied to the clipped, unabraded skin. At the completion of all exposures, the bandages were removed and the sites washed. No irritation was observed in this animal following any exposure. Based on these results, and to complete the study, 2 additional rabbits received single 4-hour exposures of the test substance. Application sites were evaluated in accordance with the method of Draize. Skin reactions for the 3-minute test site were evaluated immediately after patch removal and 1 hour later. Skin reactions for the 1-hour test site were evaluated immediately after patch removal and at approximately 24, 48, and 72 hours after removal of the 4-hour patch. Skin reactions for the 4-hour test sites were evaluated at approximately 30-60 minutes and 24, 48, and 72 hours after patch removal.

There were no deaths and no remarkable body weight changes during the study. No signs of dermal irritation were noted during the study. Based on these results, the test substance is considered to be not irritating. A classification according to Regulation (EC) No 1272/2008 is not warranted.

 

According to Regulation (EC) No 1272/2008 (CLP), section 1.1.1.3, "a weight of evidence determination means that all available information bearing on the determination of hazard is considered together, such as the results of suitable in vitro tests, relevant animal data, information from the application of the category approach (grouping, read-across), (Q)SAR results, human experience such as occupational data and data from accident databases, epidemiological and clinical studies and welldocumented case reports and observations. The quality and consistency of the data shall be given appropriate weight." In general, in vivo data from animal studies can be considered more reliable than in vitro data. The dermal irritation study from 1958 was assigned a Klimisch 4 rating as it is not sufficient for assessment of irritancy potential due to limited documentation and experimental restrictions. The most recent study (Ashland, 2013) was conducted according to OECD guideline 404 and under GLP, and it can thus be considered to be the most reliable of the studies available for assessment. The results of the study gave no indication of a skin irritation potential of the test substance.

In conclusion, evaluation of the overall evidence from in vitro and in vivo studies indicates that ammonium carbamate is not irritant to the skin.

Eye Irritation

 

Eye irritating potential of ammonium carbamate was studied in two in vitro studies and one in vivo study, performed under GLP, using OECD guideline 438, OECD guideline 437 and OECD guideline 405 protocols.

 

In the in vivo study conducted according to OECD guideline 405 (Ashland, 2013), the primary ocular irritative potential of ammonium carbamate in albino rabbits was determined. There was 1 group of 3 albino rabbits that received a single, unwashed exposure. A 100 mg dose of the test substance was instilled into the lower conjunctival sac of the right eye for each animal. The eyelid was held closed for approximately 1 second and released. The left eye of each animal was manipulated in an identical manner to simulate the dosing of the right eye and served as a contralateral control.

There were no deaths during the study. Positive corneal irritation (grades 1, 2, and 3), iridial irritation (grade 1), and conjunctival irritation (grades 2 and 3), were noted for 2 females. Positive corneal irritation was noted in 1 female throughout the study. Positive scores for iritis and/or conjunctival irritation subsided by study day 7. There were no positive effects noted in the male animal. Body weight loss was noted in one female animal but was considered to be a secondary effect in response to the ocular irritation or an aversion to handling. Based on the results from this study, the test substance was considered to cause serious damage to eyes.

In a study (BASF SE, 2010b) using fertilised chicken eggs, either a bulk volume of 25 μl (about 35 mg) per egg of the ground solid test substance, or 0.3 ml per egg of the 10% test substance solution was applied on approximately the half of chorioallantoic membrane area after careful removal of the eggshell including the inner membrane, starting from the center. The visible parts of the membrane were observed. The time until appearance of unambiguous reactions (coagulation, haemorrhaging) was recorded. Three eggs were used in each experiment. The mean time until appearance of reaction over the eggs of a treatment group was calculated (mean time to coagulation = MTC in seconds). For water-soluble test substances, a MTC < 50 s for 10% test substance solution is considered to indicate risk of serious damage to eyes. For 10% solution of ammonium carbamate, the MTC was 28 s. For the solid substance, the MTC was 31 s, in addition hemorrhaging was observed in 22 seconds. For two positive controls (0.1 M NaOH and 10% SDS) the MTC were 38 and 36 s, respectively. Based on these results, the substance was considered to be causing serious damage to eyes.

 

In the third study (BASF SE, 2010c) isolated bovine cornea was used. Each treatment group (test substance, negative and positive controls) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μl of the 10% or 20% (w/v) test-substance preparation was applied into the anterior chamber. Control tissues were concurrently treated with 750 μl of highly deionised water (negative control) or with 750 μl of 20% (w/v) solution of imidazole in highly de-ionised water (positive control). The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours. The test substance, negative and positive controls were then removed from the anterior chamber and the epithelium was washed at least 3 times before the measurement of final corneal opacity and permeability. The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). A substance is considered to elicit serious damage to eyes if the IVIS is > 55. The resulting IVIS were 113.3 and 128.7 for 10% and 20% solution of the test substance, respectively. The IVIS for the positive control was 106.4. Based on these results, ammonium carbamate is considered to elicit serious damage to eyes.

 

In addition, the eye irritation potential of the test substance in vivo was determined according to the protocols of a BASF test. One drop (ca. 50 µl) of 40% technical ammonium carbamate (no data on purity) in water was applied to the conjunctival sac of the right eye of each of two Vienna White Albino rabbits. The adjacent eye was treated with a drop of saturated NaCl solution and served as control. Eyes were not washed after instillation. The eye changes were scored several times on the treatment day, after 24 hours and 7 days afterwards, and the findings were recorded. Redness of the mucous membrane, corneal opacity, chemosis and lacrimation which lasted several days post instillation were observed in the eyes of the test animals. The effects subsided and disappeared 5-7 days post instillation (BASF AG 1958). This study is comparable to OECD guideline 438. However, it is not sufficient for the assessment of eye irritation as documentation of results is not detailed. The only score given is that for conjunctivae at the 24 hour observation time point.

 

In summary, based on overall evidence from in vitro and in vivo studies, ammonium carbamate is considered to be causing serious damage to eyes.

 


Effects on eye irritation: highly irritating

Justification for classification or non-classification

Based on overall evidence from in vitro and in vivo studies, ammonium carbamate is not considered to be irritating to skin. Based on the results of two in vitro studies, ammonium carbamate is considered to elicit serious damage to eyes. According to EU Directive 67/548/EEC, the substance should be classified as causing serious damage to eyes (Xi, R41).

In accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, the substance should be classified as Category 1, H318 (causes serious eye damage).

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