Registration Dossier

Methenamine showed some skin sensitizing potential in humans. Guinea pigs exhibited strong skin sensitization in a maximization test with a 50% aqueous solution of methenamine. In a Local Lymph Node Assay (LLNA) a positive effect concentration (EC3) of 30.6% methenamine was derived. A comparable EC3 for formaldehyde was determined in the same study. Thus, it may be concluded that also for skin sensitization formaldehyde, which may be generated by hydrolysis of methenamine in contact to skin, and not methenamine as such, is the main causative agent of sensitization.

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-05-15 to 1985-05-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Please refer to the supporting study de Jong, 2005.

Species:

guinea pig

Strain:

other: Pirbright White (Bor: DHPW)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 344-485 g
- Housing: single housing makrolon cage, type III
- Diet: standard diet ad libitum (ALTROMIN(R) Nr. 3022 from Altromin, Lage, Germany)
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Route:

other: Intradermal (Day 1), Epidermal (Day 8)

Vehicle:

physiological saline

Concentration / amount:

Induction:
Intrademal 30 %
Epidermal: paste (0.5 g test substance with 0.4 ml physiol. saline)

Challenge: 50 %

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction:
Intrademal 30 %
Epidermal: paste (0.5 g test substance with 0.4 ml physiol. saline)

Challenge: 50 %

No. of animals per dose:

control group: 10
test group: 20

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
Introdermal
- No. of exposures: 6 (2 x test substance, 2 x test substance / FCA (1:1), 2x FCA)
- Exposure period: Day 1
- Test groups: test group: see above
- Control group: same treatment, but vehicle instead of test substance
- Site: shoulder region
- Frequency of applications: once on day 1
- Duration: single exposure, next exposure on day 8
- Concentrations: 30 %

Epidermal
- No. of exposures: 1
- Exposure period: Day 8
- Test groups: occlusive patch with test substance: paste (0.5 g test substance with 0.4 ml physiol. saline)
- Control group: same treatment, but vehicle instead of test substance
- Site: shoulder region
- Frequency of applications: once on day 8
- Duration: 48 h
- Concentrations: paste (0.5 g test substance with 0.4 ml physiol. saline)

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: day 22, day 23
- Exposure period: 24 h
- Test groups: right flank (vehicle), left flank (test substance)
- Control group: same as test group
- Site: flank
- Concentrations: 50 %
- Evaluation (hr after challenge): 24 and 48 hrs after last challenge

Challenge controls:

yes (see details on study design)

Positive control substance(s):

no

Positive control results:

no positive control

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

not tested

Clinical observations:

not tested

Remarks on result:

not measured/tested

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

15

Total no. in group:

20

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

15

Total no. in group:

20

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

not tested

Clinical observations:

not tested

Remarks on result:

not measured/tested

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Methenamine is sensitising in the guinea pig maximation test.

Executive summary:

In a dermal sensitization study (GPMT) with methenamine (> 99 %) in physiological saline 6-9 weeks old female Pirbright White guinea gigs were tested using the method of Magnusson and Kligman.

No clinical signs were noted. After intradermal (30 %) and epidermal induction (paste: 0.5 g test substance in 0.4 ml physiol. saline) and challenge (50 %) 15 out of 20 animals showed etythema and edema 24 and 48 h after the last challenge. In the negative control group none of the 10 animals tested showed signs of sensitisation.

In this study, methenamine is a dermal sensitizer.

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study:

In a dermal sensitization study (GPMT) with methenamine (> 99 %) in physiological saline 6-9 weeks old female Pirbright White guinea gigs were tested using the method of Magnusson and Kligman.

No clinical signs were noted. After intradermal and epidermal induction (30 %) and challenge (50 %) 15 out of 20 animals showed etythema and edema 24 and 48 h after the last challenge. In the negative control group none of the 10 animals tested showed signs of sensitisation.

In this study, methenamine is a dermal sensitizer.

Supporting study:

In a Local Lymph Node Assay (LLNA) similar to OECD Guideline 429 (de Jong, 2005), an EC3 (test concentration, which causes a Stimulation Index (SI) of the auricular lymph nodes of 3.0) of 30.6% was determined for methenamine, while an EC3 as low as 0.96% was determined for formaldehyde in the same study. The test was performed treating young adult (6-8 weeks of age) female BALB/c mice with methenamine dissolved in a acetone-olive oil mixture (4:1). Test concentrations were 2.5, 5, 10 and 20%. In order to enhance possible low responses of weak sensitizers animals were pretreated with sodium dodecyl sulfate 1% (SDS) on the dorsum of the ears one hour before administration. In second test with repeated exposures the fist test was coonfirmed revealing a Stimulation Index of 5.78. Results of the LLNA affirm the classification of methenamine as a skin sensitizer.

Human data:

In 1992/1993 among 2150 patients with dermatitis tested for methenamine sensitivity (1% in petrolatum) 27 (1.3%) showed a positive skin reaction. Out of these, 15 (0.7%) were interpreted as irritant reaction and 12 patients (0.5%) were regarded as sensitized. In 1994/1995, among 3082 persons tested, 38 (1.2%) showed positive skin reactions with methenamine Out of these, 15 (0.5%) were interpreted as irritant reactions and 23 patients (0.8%) were regarded as sensitized (IVDK, 1996).

Within a study related to rubber-related allergic contact dermatitis, patch-testing was performed with a number of rubber-related substances. Out of 309 patients, 1.9% showed positive skin reactions with 2% methenamine (Holness and Nethercott, 1997).

A 54-year-old employee with an itchy eruption on his hands, neck and shoulders was presented in March 1987. He had worked in a foundry for 10 years being engaged in a core molding process and had developed itchy eruptions on the dorsum of his hands, neck and shoulders 3 years ago. The eruptions and itching were aggravated by sweating. His work was making a mixture of sand, phenolic resin, methenamine and lubricant, and he was often covered with the mixture. Offensive stench gas was evaporated from the heated sand core. Open patch testing and closed patch testing with raw materials and chemicals detected within the gas from the heated sand core were performed. The patient reacted positive to 1% methenamine in petrolatum, but did not react positively to formaldehyde. Repeated patch testing in October 1987 confirmed the negative reaction to formaldehyde (Hayakawa et al., 1988).

In one molding resin producing factory, 10% of the workers were affected with dermatitis during a period of one year. Patch tests with various types of resins performed on 10 cases showed that sensitivity to methenamine and to formaldehyde was the cause of 80% of the dermatitis in this plant (Schwartz, 1957).

Methenamine adversely affected 60 employees in a local rubber factory. Acute dermatitis of the exposed surfaces was the principle symptom: Removal of methenamine from the entire rubber stock alone prevented further cases (Cronin, 1924). However, it cannot be concluded from this case study that dermal irritation was caused by sensitization.

The total number of cases observed in a report on the shellac and lacquer industries amounted to 7 patients each, all of whom manifested allergic symptomatology upon separate exposure to ethylenediamine and methenamine. A skin test with 1% dilutions gave severe positive reactions, characterized by immediate wheal formation. The provocative inhalation test with methenamine was positive in 7/7 patients. On exposure to the substance, the patients showed either wheezing and heaviness on the chest or severe asthma, allergic coryza, or skin manifestations of allergy. 14 patients from the beauty industry were also challenged with methenamine and other substances. Of these, 13 showed diverse positive responses. However, since there was exposure to several chemicals, cross-reactivity may have occurred. In a factory where machines for the processing of chemicals are produced, a worker who tested a machine with chemicals used for the preparation of rubber was exposed directly after the chemicals had run through the machine. He suffered from coughing and wheezing resulting in three consecutive severe asthma attacks after repeated exposure (Gelfand, 1963). Due to the study limitations (only a few case reports, no detailed description of the exposure level of the individuals affected, and possible contributions of other substances in addition to methenamine to positive findings) no clear conclusion on respiratory sensitization can be drawn from this study.

A more recent study was designed to assess the health effects of methenamine on the airways and the skin of workers in the chemical industry (Merget et al., 1999). 17 “highly” exposed and 16 “low” exposed employees of a methenamine producing plant were examined (a quantification of high or low exposure is not given). Among the 17 highly exposed persons 8 subjects were baggers, 5 were shift leaders and 4 were executive staff. 11 blue collar and 5 white collar workers with no exposure to known sensitizers were recruited as controls.(n = 16). Four persons, who had left the production department and another 15 who had left the plant for reasons not related to methenamine exposure, were also included (n = 19). Medical history, total and specific IgE to four environmental allergens, lung function and bronchial responsiveness to metacholine were assessed by standard procedures. Lung function was evaluated by measurement of forced expiratory volume in 1s and maximal expiratory flow at 50% of forced vital capacity (pneumotachograph, CustoVit, CustoMed, Munich, Germany). Measuring conditions and reference values were chosen as described recently (Quanjer et al., 1993). Bronchial hyperresponsiveness was assessed as described in Merget et al. (1996) with two modifications: methacholine was used instead of histamine and maximally 31 instead of 63 cumulative breaths of the 8 mg/mL methacholine solution were administered. Skin prick tests were performed with known sensitizers and methenamine. 64.7 % of the exposed subjects and 68.8 % of the controls reported symptoms during the year before the study began, most of them not related to work. Work-related symptoms and objective parameters did not show differences between groups, and skin tests showed no sensitization to methenamine. Among those who had left methenamine production for medical reasons, two former baggers showed sensitization to methenamine by patch test and reported eczema during exposure which disappeared after removal from exposure. Geometric mean methenamine concentrations were 0.3 mg/m3 in shiftleaders and 0.6 mg/m3 in baggers. It was concluded that high exposures to methenamine may cause allergic contact dermatitis. However, there was no evidence of an increased risk for occupational asthma at airborne concentrations in the range of 0.2 - 2.6 mg/m3 (Merget et al., 1999).

Endpoint conclusion:

no study available

Additional information:

Human data:

The total number of cases observed in a report on the shellac and lacquer industries amounted to 7 patients each, all of whom manifested allergic symptomatology upon separate exposure to ethylenediamine and methenamine. A skin test with 1% dilutions gave severe positive reactions, characterized by immediate wheal formation. The provocative inhalation test with methenamine was positive in 7/7 patients. On exposure to the substance, the patients showed either wheezing and heaviness on the chest or severe asthma, allergic coryza, or skin manifestations of allergy. 14 patients from the beauty industry were also challenged with methenamine and other substances. Of these, 13 showed diverse positive responses. However, since there was exposure to several chemicals, cross-reactivity may have occurred. In a factory where machines for the processing of chemicals are produced, a worker who tested a machine with chemicals used for the preparation of rubber was exposed directly after the chemicals had run through the machine. He suffered from coughing and wheezing resulting in three consecutive severe asthma attacks after repeated exposure (Gelfand, 1963). Due to the study limitations (only a few case reports, no detailed description of the exposure level of the individuals affected, and possible contributions of other substances in addition to methenamine to positive findings) no clear conclusion on respiratory sensitization can be drawn from this study.

A more recent study was designed to assess the health effects of methenamine on the airways and the skin of workers in the chemical industry (Merget et al., 1999). 17 “highly” exposed and 16 “low” exposed employees of a methenamine producing plant were examined (a quantification of high or low exposure is not given). Among the 17 highly exposed persons 8 subjects were baggers, 5 were shift leaders and 4 were executive staff. 11 blue collar and 5 white collar workers with no exposure to known sensitizers were recruited as controls.(n = 16). Four persons, who had left the production department and another 15 who had left the plant for reasons not related to methenamine exposure, were also included (n = 19). Medical history, total and specific IgE to four environmental allergens, lung function and bronchial responsiveness to metacholine were assessed by standard procedures. Lung function was evaluated by measurement of forced expiratory volume in 1s and maximal expiratory flow at 50% of forced vital capacity (pneumotachograph, CustoVit, CustoMed, Munich, Germany). Measuring conditions and reference values were chosen as described recently (Quanjer et al., 1993). Bronchial hyperresponsiveness was assessed as described in Merget et al. (1996) with two modifications: methacholine was used instead of histamine and maximally 31 instead of 63 cumulative breaths of the 8 mg/mL methacholine solution were administered. Skin prick tests were performed with known sensitizers and methenamine. 64.7 % of the exposed subjects and 68.8 % of the controls reported symptoms during the year before the study began, most of them not related to work. Work-related symptoms and objective parameters did not show differences between groups, and skin tests showed no sensitization to methenamine. Among those who had left methenamine production for medical reasons, two former baggers showed sensitization to methenamine by patch test and reported eczema during exposure which disappeared after removal from exposure. Geometric mean methenamine concentrations were 0.3 mg/m3 in shiftleaders and 0.6 mg/m3 in baggers. It was concluded that high exposures to methenamine may cause allergic contact dermatitis. However, there was no evidence of an increased risk for occupational asthma at airborne concentrations in the range of 0.2 - 2.6 mg/m3 (Merget et al., 1999).

Guinea pigs exhibited strong skin sensitization in a maximization test with a 50% aqueous substance solution. In a Local Lymph Node Assay (LLNA) a positive effect concentration (EC3) of 30.6% methenamine was derived. A comparable EC3 for formaldehyde was determined in the same study. Thus, it may be concluded that also for skin sensitization formaldehyde, which may be generated by hydrolysis of methenamine in contact to skin, and not methenamine, is the main causative agent of sensitization. Nevertheless, taken all data together, the existing classification with R 43 "May cause sensitization by skin contact" according to 67/548/EEC and skin sens. cat.1B (H317) according to CLP (GHS) is warranted and justified. This C+L proposal is in line with occupational findings as Methenamine has demonstrated some skin sensitizing potential in humans.

From earlier studies a number of cases of allergic symptoms such as wheezing and asthma were reported upon inhalation exposure to methenamine (Gelfand, 1963). Some other occupational reports of a potental respiratory sensitisation potential were not confirmed in well documented newer studies. In consequence, there is not clear evidence of a respiratory sensitization potential of methenamine at airborne levels present at workplaces. Classification with R 42 „May cause sensitization by inhalation“ according to 67/548/EEC and resp. sens. cat.1 (H334) according to CLP (GHS) is therefore not justified.