Registration Dossier

Administrative data

Description of key information

There is sufficient evidence for sensitizing properties of formaldehyde in the guinea pig maximisation test (GPMT), in the Buehler test in guinea pigs and in the mouse local lymph node assay (LLNA). Formaldehyde is also a dermal allergen in humans. Although rare, anaphylaxis has been documented in case reports.
Animal studies do not indicate that formaldehyde may induce sensitization to the respiratory tract. A very limited number of case reports have been published on formaldehyde-related asthma but these data do not provide sufficient evidence that formaldehyde should be considered a risk factor for respiratory tract sensitization.
Recently the data on skin and respiratory tract sensitization by formaldehyde have been comprehensively reviewed by the German MAK commission (MAK, 2010). With regard to skin sensitization MAK (2010) concluded that allergic contact dermatitis is relatively frequently observed in patients by diagnostic patch testing. In addition, many experimental animal studies gave positive results for sin sensitization. As asthma related to formaldehyde is concerned, MAK (2010) concluded that the allergological findings do not provide a consistent pattern. In inhalation challenge tests generally immediate reactions were observed, dual or late reactions were rare. A differentiation against irritation often is difficult and specific IgE antibodies, if found, mostly did not correlate with the respiratory symptoms. Overall, a relationship of respiratory symptoms with formaldehyde is only in few cases sufficiently documented. The small number of reliable findings in comparison to the broad exposure potential would not warrant to classify formaldehyde as a respiratory sensitizer according to the criteria of the MAK commission.

Specialized studies with experimental animals did not provide evidence for a formaldehyde-induced respiratory allergy. In general, the results in human studies did not provide clear evidence for a formaldehyde-induced respiratory allergy. Reported symptoms in case reports might also be related to irritant effects.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
comparable with guideline 429; no details about the test substance, no data about body weight and irritating effects, animals sacrificed at day 5 instead of day 6
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(Animals sacrificed at day 5 instead of day 6)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Formalin, source Fisons, Loughborough, UK (37% aqueous solution of formaldehyde, presumably in 10% methanol)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
- Source: Harlan Olac Ltd., Bicester, Oxfordshire
- Age/weight at study initiation: 6-12 weeks-old/ no data
no further details
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Vehicle control (4:1 acetone/olive oil), 0.1, 0.5, 1, 5, 10 % Formalin
No. of animals per dose:
4 (control n=4)
Details on study design:
Topical exposure to the allergen (25 µL of the test substance solution at various formaldehyde concentrations applied to the dorsum of each ear, daily applications on 3 consecutive days) causes lymphocyte activation in the lymph nodes draining the site of application; proliferation of lymph node cells determined after labelling of cells with 3H-methyl thymidine (i.v. injection of 3H-thymidine 5 days after exposure initiation and mice sacrificed 5 h later). Lymph nodes excised 5 h after i.v. injection of labelled thymidine and lymph nodes pooled for each dose group. Incorporation of 3H-thymidin measured via ß-scintilation counting.
Measured parameter: Mean dpm/pooled nodes
Positive control substance(s):
other: In parallel experiments other aldehyde tested with positive results.
Statistics:
no
Parameter:
SI
Value:
>= 3
Test group / Remarks:
1, 5, 10%
Parameter:
other: disintegrations per minute (DPM)
Test group / Remarks:
0.5, 1, 5, 10%
Remarks on result:
other: Increase in 3H-thymidine incorporation at ≥ 0.5% formalin (details in Table below).
Parameter:
EC3
Value:
0.35
Remarks on result:
other: the EC3 was 0.93% formalin or 0.35% formaldehyde

Concentration of the test substance formalin in % (w/v)

Incorporation of 3H-thymidin in lymph node cells measured in dpm/pooled lymph nodes

Stimulation index (SI) of test compared to vehicle control

Vehicle control

611

1

0.1

594

0.97

0.5

1165

1.91

1

1940

3.17

5

3197

5.23

10

5280

8.59

Concentration related to formalin

Increase in 3H-thymidine incorporation at ≥ 0.5% formalin. The EC3value (3-fold stimulation of proliferation as an index of the relative potency of a contact allergen) was 0.93% formalin or 0.35% formaldehyde.

Interpretation of results:
other: sensitising
Conclusions:
These results suggested skin-sensitizing activity of the test substance in the LLNA.
Executive summary:

The study is comparable with OECD guideline 429 with acceptable restrictions (no details about the test substance, no data about body weight and irritating effects, animals sacrificed at day 5 instead of day 6).

In this Local Lymph Node Assay (LLNA) 4 mice per dose received 25 µL of the test substance solution at various formalin (37%) concentrations (0, 0.1, 0.5, 1, 5, 10 % corresponding to 0, 0.04, 0.2, 0.4, 1.9, 3.7% formaldehyde solution) applied to the dorsum of each ear (daily applications on 3 consecutive days); after labelling of cells with 3H-methyl thymidine (i.v. injection of 3H-thymidine 5 days after exposure initiation and mice sacrificed 5 h later) lymph nodes were excised 5 h after i.v. injection of labelled thymidine and lymph nodes pooled for each dose group.

The stimulation index increased dose dependently. No induction was detected at 0.04% formaldehyde and first sensitizing effects at 0.2%.

The EC3value (3-fold stimulation of proliferation as an index of the relative potency of a contact allergen) was 0.93% formalin or 0.35% formaldehyde.

Conclusion: Formaldehyde is sensitizing in the LLNA.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no data on positive controls [but clearly positive results]; no data on impurities of the test substance; no details about the pretest; challenge in controls using patches without formaldehyde resulted in some animals in score 1 erythema; high concentration for intradermal induction
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Pt.-Nr.: Z 69099
stability was checked
formaldehyde in aqueous solution (no data about concentration of stock solution)
No further data
Species:
guinea pig
Strain:
other: DHPW
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen (Ger)
- Age at study initiation: no data
- Weight at study initiation: initial body weight range 270-360 g)
- Housing: 5 per cage
- certified diet and tap water ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25
- Humidity (%): 55-80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
5%, 0.1 mL (intradermal); 5%, 0.5 mL (epicutaneous)
Day(s)/duration:
Epicutaneous: 1 week after intradermal induction, duration 48h
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
2%
Day(s)/duration:
3 weeks after intradermal induction, duration 24h
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
0.5%
Day(s)/duration:
5 weeks after intradermal inducation, duration 24h
No. of animals per dose:
10 controls for the 1st challenge and 10 controls for the 2nd challenge
20 test animals
Details on study design:
Induction
1) 2 intradermal injections of 0.1 mL of 5% test substance solution with and without FCA plus 50% FCA alone (totally 3 x 2 injections) 24 h after shaving; same procedure in controls but without test substance.
2) 1 week after intradermal injection dermal application of 5% test substance solution for 48 h (0.5 mL on a patch of 2 x 4 cm²; occlusive; patch covering injection sites; skin shaved 24 h prior to topical application) for induction; shame exposure of controls without test substance; patch removal after 48 h.

1st Challenge
3 weeks after intradermal injection topical application of a patch soaked with 2% aqueous solution of formaldehyde (no data about patch size; occlusive; 24 h exposure period; animals shaved and depilated 24 h prior to treatment); control patches without test substance (but vehicle) on the other flank.
readings 24 and 48 h after patch removal

2nd challenge
5 weeks after intradermal injection topical application of a patch soaked with 0.5% aqueous solution of formaldehyde (no data about patch size; occlusive; 24 h exposure period; animals shaved and depilated 24 h prior to treatment); control patches without test substance (but vehicle) on the other flank.
readings 24 and 48 h after patch removal

Scoring system according to the OECD Guideline 406 (score 0, 1, 2, or 3 for erythema)
Challenge controls:
Also treatment with the same concentrations used for challenge in test animals (test and control patches)
Positive control substance(s):
not specified
Statistics:
No data
Positive control results:
No data
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
effects different from control patches
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 6.0. Total no. in groups: 20.0. Clinical observations: effects different from control patches.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
effects different from control patches
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: effects different from control patches.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
2%
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
effects different from control pathches
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2%. No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: effects different from control pathches.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction different from control patch
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 2%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction different from control patch.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.5%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
effects different from control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5%. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: effects different from control patches.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects different from control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects different from control patches.
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
0.5%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
effects different from control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: test group. Dose level: 0.5%. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: effects different from control patches.
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects different from control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: negative control. Dose level: 0.5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects different from control patches.
Interpretation of results:
other: sensitising
Conclusions:
Sensitizing effects in the GPMT.
Executive summary:

Comparable to Guideline study with acceptable restrictions (no data on positive controls [but clearly positive results]; no data on impurities of the test substance; no details about the pretest; challenge in controls using patches without formaldehyde resulted in some animals in score 1 erythema; high concentration for intradermal induction).

In the guinea pig maximization test 20 males were induced by intradermal injection and topical application (1 week after intradermal induction) with 5% formaldehyde, 20 male controls were shame treated (10 control animals for each challenge). First challenge with 2% formaldehyde solution was performed 3 weeks after intradermal induction in test animals and controls; controls gave valid results 48 and 72 h after application of challenge patch (no effects different from control patches without test substance). In test animals 2% formaldehyde resulted in positive reaction in 6/20 (1st reading 48 h after application) and 8/20 (2nd reading, 72 h) tested animals. For the second challenge 5 weeks after intradermal induction 0.5% formaldehyde solution was used. Valid results were obtained with controls (no effects different from control patches without formaldehyde); 10/20 and 7/20 test animals showed positive reactions different from control patches 48 and 72 h after application of the challenge patch, respectively.

Conclusion: Sensitizing effects in the GPMT.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no data on positive controls [but clearly positive results]; no data on impurities of the test substance; no details about the pretest; high concentration used for intradermal induction
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Remarks:
(Bayer AG, Institute of Toxicology)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Pt.-Nr.: Z 69099
stability was checked; homogeneity was given
37% formaldehyde in aqueous solution (acid-free)
stored in the dark at 20-23°C
No further data
Species:
guinea pig
Strain:
other: DHPW
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen (Ger)
- Age at study initiation: 5-7 weeks
- Weight at study initiation: initial body weight range 281-357 g
- Housing: 5 per cage
- certified diet and tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
5%, 0.1 mL (intradermal); 5%, 0.5 mL (epicutaneous)
Day(s)/duration:
Epicutaneous: 1 week after intradermal inducation, duration 48 h
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
0.5 mL, 2%
Day(s)/duration:
3 weeks after intradermal induction, duration 24h
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
0.5 mL, 0.2%
Day(s)/duration:
5 weeks after intradermal induction, duration 24h
No. of animals per dose:
10 controls for the 1st challenge and 10 controls for the 2nd challenge
20 test animals
Details on study design:
Induction
1) 2 intradermal injections of 0.1 mL of 5% test substance solution with and without FCA plus 50% FCA alone (totally 3 x 2 injections) 24 h after shaving; same procedure in controls but without test substance.
2) 1 week after intradermal injection dermal application of 5% test substance solution for 48 h (0.5 mL on a hypo-allergenic patch of 2 x 4 cm²; occlusive; patch covering injection sites; skin shaved 24 h prior to topical application) for induction; shame exposure of controls without test substance; patch removal after 48 h.

1st Challenge
3 weeks after intradermal injection topical application of a patch soaked with 0.5 mL of 2% aqueous solution of formaldehyde (no data about patch size; occlusive; 24 h exposure period; animals shaved and depilated 24 h prior to treatment); control patches without test substance (but 0.5 mL vehicle) on the other flank.
readings 24 and 48 h after patch removal

2nd challenge
5 weeks after intradermal injection topical application of a patch soaked with 0.5 mL 0.2% aqueous solution of formaldehyde (no data about patch size; occlusive; 24 h exposure period; animals shaved and depilated 24 h prior to treatment); control patches without test substance (but 0.5 mL vehicle) on the other flank.
readings 24 and 48 h after patch removal

Scoring system according to the OECD Guideline 406 (score 0, 1, 2, or 3 for erythema)
Clinical signs recorded daily, body weight determined weekly and day 24 & 38
Necropsy of animals found dead (or moribund)
Challenge controls:
Also treatment with the same concentrations used for challenge in test animals (test and control patches)
Positive control substance(s):
not specified
Statistics:
No data
Positive control results:
No data
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
9
Total no. in group:
18
Clinical observations:
no effects of control patches
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 9.0. Total no. in groups: 18.0. Clinical observations: no effects of control patches.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects of control patches
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects of control patches.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
2%
No. with + reactions:
6
Total no. in group:
18
Clinical observations:
no effects of control patches
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2%. No with. + reactions: 6.0. Total no. in groups: 18.0. Clinical observations: no effects of control patches.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction of control patch
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 2%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction of control patch.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0.2%
No. with + reactions:
2
Total no. in group:
18
Clinical observations:
no effects of control patches; 1 animals with eschar formation
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0.2%. No with. + reactions: 2.0. Total no. in groups: 18.0. Clinical observations: no effects of control patches; 1 animals with eschar formation .
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0.2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects of control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.2%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects of control patches.
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
0.2%
No. with + reactions:
3
Total no. in group:
18
Clinical observations:
no effects of control patches; eschar formation in all 3 positive animals
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: test group. Dose level: 0.2%. No with. + reactions: 3.0. Total no. in groups: 18.0. Clinical observations: no effects of control patches; eschar formation in all 3 positive animals.
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
0.2%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects of control patches
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 72.0. Group: negative control. Dose level: 0.2%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects of control patches.
Interpretation of results:
other: sensitising
Conclusions:
Sensitizing effects in the GPMT.
Executive summary:

Guideline study with acceptable restrictions (no data on positive controls [but clearly positive results]; no data on impurities of the test substance; no details about the pretest; high concentration used for intradermal induction).

In the guinea pig maximization test 20 males were induced by intradermal injection and topical application (1 week after intradermal induction) with 5% formaldehyde, 20 male controls were shame treated (10 control animals for each challenge). Two animals of the treatment group died during the experiment but death was not treatment related. First challenge with 2% formaldehyde solution was performed 3 weeks after intradermal induction in test animals and controls; controls gave valid results 48 and 72 h after application of challenge patch (no effects at all). In test animals 2% formaldehyde resulted in positive reaction in 9/18 (1st reading 48 h after application) and 6/18 (2nd reading, 72 h) tested animals. For the second challenge 5 weeks after intradermal induction 0.2% formaldehyde solution was used. Valid results were obtained with controls (no effects); 2/18 and 3/18 test animals showed positive reactions 48 and 72 h after application of the challenge patch, respectively.

Conclusion: Sensitizing effects in the GPMT.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
partly limited documentation, e.g. no details on pretest and positive control
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Formaldehyde were obtained from Brunschig Chemie Amsterdam, the Netherlands.
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
6-8 weeks old, bred under SOF conditions
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.06, 0.23, 0.92, 1.85%
No. of animals per dose:
Control: n=6, tested concentration: n=3
Parameter:
SI
Value:
1
Test group / Remarks:
Control
Parameter:
SI
Value:
1
Test group / Remarks:
0.06%
Parameter:
SI
Value:
1.4
Test group / Remarks:
0.23%
Parameter:
SI
Value:
3.8
Test group / Remarks:
0.92%
Parameter:
SI
Value:
7
Test group / Remarks:
1.85%
Parameter:
EC3
Value:
0.96
Remarks on result:
other: L05: 0.75 - L95: 1.32) 90% confidence limit

EC3: 0.96% (L05: 0.75 - L95:1.32; 90% confidence limit)

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
EC3 of 0.96% (corresponding to 240 µg/cm²) was reported
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
partly limited documentation, e.g. no details about results or no data about purity of the test substance; no details on pretest and positive control; low number of animals but clearly positive results
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
not specified
Type of study:
Buehler test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Buehler test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Formalin (37% formaldehyde), source Sigma Chemicals, St Louis, USA (presumably aqueous solution of formaldehyde in 10% methanol).
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
Source: Harlan Olac, Oxon, UK
Age/weight at study initiation: No data / ca. 350 g
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5%, duration of 6 hours
Day(s)/duration:
this procedure was repeated once weekly for 3 consecutive weeks
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
1%, duration of 6 hours
Day(s)/duration:
12-14 days after last induction
No. of animals per dose:
10 (control n=5)
Details on study design:
- Induction schedule
topical (occlusive); filter paper patch saturated with 5% test substance for 6 h on the shaved skin under occlusive dressing
this procedure was repeated once weekly for 3 consecutive weeks
Controls shame-treated

- Challenge schedule:
12-14 days after the last induction patch with 1% test substance under occlusive dressing for 6 h (shaved skin); reading according to OECD406 was performed 24 h after patch removal.

- Positive control substance: No data (but positive results)
- Pilot study: Irritant concentration threshold determined. No details of this test given.
Challenge controls:
5 control animals received the test substance using the same concentration than in test animals
Positive control substance(s):
not specified
Statistics:
No data
Reading:
1st reading
Hours after challenge:
30
Group:
test group
Dose level:
1%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
no details available
Reading:
1st reading
Hours after challenge:
30
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5

2nd reading not performed (clearly positive)

Interpretation of results:
other: sensitising
Conclusions:
Formalin was sensitizing in the Buehler test
Executive summary:

Comparable to guideline study with acceptable restrictions (partly limited documentation, e.g. no details about results or no data about purity of the test substance; no details on pretest and positive control; low number of animals but clearly positive results).

In the Buehler test 10 guinea pigs were induced by topical application of 5% formaldehyde solution for 6 h under occlusive dressing; this procedure was repeated once weekly for 3 consecutive weeks. Five controls were shame-treated. The challenge (6 h occlusive patch with 1% test substance) was performed 12 -14 days after the last induction. A positive reaction was found in 7/10 animals. No positive reaction was detected in 5 negative controls.

Under the condition of this assay sensitizing effects were recorded in the Buehler test.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
partly limited documentation, e.g. no data about purity of the test substance; no details on pretest and positive control
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Study according to Magnusson & Kligman, Allergic contact dermatitis in the Guinea Pig, Charles C Thomas, Springfield, IL, USA
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Formalin (37% formaldehyde), source Sigma Chemicals, St Louis, USA (presumably aqueous solution of formaldehyde in 10% methanol).
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
Source: Harlan Olac, Oxon, UK
Age/weight at study initiation: No data / ca. 350 g
Number of animals per group: 10 (control n=5)
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
0.25% (intradermal), 10% (epicutaneous)
Day(s)/duration:
Epicutaneous: 6-8 days later than injections, duration 48h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
2%
Day(s)/duration:
12-14 days after last induction, duration 24h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (control n=5)
Details on study design:
- Induction schedule
6 intradermal injections of 0.25% formaldehyde solution (presumably with and without FCA; no details) followed by dermal application of 10% test substance solution for 48 h (occlusive) 6-8 days later (no further details).
- Way of Induction: Intradermal & topical (occlusive)
- Concentrations used for induction
intradermal injections of 0.25% formaldehyde solution. Dermal 10% test substance (which should cause mild to moderate irritation according to guideline)
- Concentration Freunds Complete Adjuvant (FCA): No data
- Challenge schedule: One challenge 12-14 days after last induction; concentrations used for challenge: 2% in physiological saline, 24 h occlusive patch (maximum non-irritant concentration); no rechallenge
- Scoring schedule: Similar to OECD 406; 24 h after removal of the patch
- Removal of the test substance: after 24 h
- Positive control substance: No data (but positive results)
- Pilot study: Irritant concentration threshold determined. No details of this test given.
Challenge controls:
5 control animals received the test substance using the same concentration than in test animals
Positive control substance(s):
yes
Remarks:
(no details)
Statistics:
No data
Positive control results:
Valid, but no details given
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
10
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
5

Results of pilot studies

No irritant effects at 2% test substance in physiological saline

2nd reading after 72 h not performed (clearly positive)

Interpretation of results:
other: sensitising
Conclusions:
Formalin was sensitizing in the GPMT.
Executive summary:

The study is comparable to a guideline study with acceptable restrictions (partly limited documentation, e.g. no data about purity of the test substance; no details on pretest and positive control).

In this Guinea pig maximization test 10 animals were induced by intradermal injection with 0.25% formaldehyde and epicutaneous applications of 10% solution. A pilot study was conducted. For occlusive challenge 2% solution was used. A positive reaction was found in 10/10 animals. No positive reaction was detected in 5 negative controls.

Under the condition of this assay sensitizing effects were recorded in the GPMT.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
partly limited documentation, e.g. no details on pretest and positive control
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Formalin (37% formaldeyhde)
Species:
mouse
Strain:
other: CRA/Ca
Sex:
female
Details on test animals and environmental conditions:
6-12 weeks old
Vehicle:
other: acetone and DMF
Concentration:
25 µL of 0.25, 0.5, 1, 2.5, and 5%
No. of animals per dose:
4
Parameter:
EC3
Value:
0.54
Test group / Remarks:
Vehicle: Acetone
Remarks on result:
other: Acetone: 0.180 mol/L (corresponding to 0.54%)
Parameter:
EC3
Value:
0.33
Test group / Remarks:
Vehicle: DMF
Remarks on result:
other: DMF: 0.110 mol/L (corresponding to 0.33%)
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
EC3 values were 0.180 mol/L for acetone and 0.110 mol/L for DMF
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no data on positive controls [but clearly positive results]; no data on impurities of the test substance (but analytical grade]; high concentration for intradermal induction
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Remarks:
(Hoechst AG, Dept. of Toxicology)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Source: Riedel del Haen (Ger)
analytical grade
35% formaldehyde in aqueous solution
No further data
Species:
guinea pig
Strain:
other: Pirbright white [Hoe: DHPK(SPFLac)]
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF-Zucht
- Age at study initiation: no data
- Weight at study initiation: initial mean body weight: 247 g (range: 219-279 g)
- Housing: 5 per cage
- certified diet and tap water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
5%, 0.1 mL (intradermal); 5%, 0.5 mL (epicutaneous)
Day(s)/duration:
Intradermal: at day 1. Epicutaneous: at day 9, duration 48h
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
4% (left flank) and 2% (right flank), 0.5 mL
Day(s)/duration:
Day 22, duration 24h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
4% (left flank) and 2% (right flank), 0.5 mL
Day(s)/duration:
Day 36, duration 24h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 controls
20 test animals
Details on study design:
Induction
1) 2 intradermal injections of 0.1 mL of 5% test substance solution with and without FCA plus 50% FCA alone (totally 3 x 2 injections) after shaving at day 1 for induction (same procedure in controls but without test substance)
2) day 9 dermal application of 5% test substance solution for 48 h (0.5 mL on a patch of 2 x 4 cm²; occlusive; patch covering injection sites) for induction at day 9-11 (shame exposure of controls without test substance); patch removal after 48 h.

1st Challenge
Day 22 left flank topical application of 0.5 mL 4% formaldehyde in phys. saline (2 x 2 cm² patch; occlusive; 24 h exposure period)
and
Day 22 right flank topical application of 0.5 mL 2% formaldehyde in phys. saline (2 x 2 cm² patch; occlusive; 24 h exposure period)
Day 23 patch removal
Day 24 and 25 readings

2nd challenge
Day 36 same procedure than day 22-24 (one reading 24 h after patch removal)

Scoring system according to the OECD Guideline 404
Challenge controls:
Also treatment with the same concentrations used for challenge in test animals
Positive control substance(s):
not specified
Statistics:
No data
Positive control results:
No data
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
4%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
score 1 erythema in 2 animals (other score 2-3)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 4%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: score 1 erythema in 2 animals (other score 2-3).
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
4%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 4%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no reaction .
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
4%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
5/20 score 1 erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 4%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: 5/20 score 1 erythema.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
4%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
only score 1 in 2 animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 4%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: only score 1 in 2 animals.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
score 1 in 11/20 animals (other animals score 2)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: score 1 in 11/20 animals (other animals score 2).
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
2%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
score 1 in 13 animals (other score 2-3)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2%. No with. + reactions: 18.0. Total no. in groups: 20.0. Clinical observations: score 1 in 13 animals (other score 2-3).
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
2%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
score 1
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 2%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: score 1.
Interpretation of results:
other: sensitising
Conclusions:
Sensitizing effects in the GPMT.
Executive summary:

Guideline study with acceptable restrictions (no data on positive controls [but clearly positive results]; no data on impurities of the test substance (but analytical grade]; high concentration for intradermal induction).

In the guinea pig maximization test 20 females were induced by intradermal injection and topical application with 5% formaldehyde, 10 female controls were shame treated. Challenge was performed with 2 or 4% solution in test animals and controls; controls gave valid results after both concentrations. In test animals 4% formaldehyde resulted in positive reaction in all tested animals and 2% test substance in 80% positive reactions at the 1st reading.

Conclusion: Sensitizing effects in the GPMT.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
low number of negative controls; partly limited documentation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
(low number of control animals)
Principles of method if other than guideline:
Study according to Magnusson & Kligman, Allergic contact dermatitis in the Guinea Pig, Charles C Thomas, Springfield, IL, USA
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Formalin, source BDH Ltd. (presumably aqueous solution of formaldehyde in 10% methanol); no further data
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
300-350 g body weight at initiation
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
0.25% (intradermal), 10% (epicutaneous)
Day(s)/duration:
Epicutaneous 6-8 days later than intradermal, duration 48h
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
2%
Day(s)/duration:
12-14 days after last induction, 24h
No. of animals per dose:
10 exposed; 4 controls
Details on study design:
- Pretest performed on irritant effects: Irritation tests performed to determine appropriate concentrations of formaldehyde for induction (mildly irritant concentration) and challenge (maximum non-irritant concentration). No details given.
- Induction schedule
6 intradermal injections of 0.25% test substance solution with and without FCA followed by dermal application of 10% test substance solution for 48 h (occlusive) 6-8 days later (no further details).
Concentrations used for induction: Dermal 10% test substance (which should cause mild to moderate irritation according to guideline)
- Challenge schedule
One challenge 12-14 days after last induction.
Concentrations used for challenge: 2% in physiological saline, 24 h occlusive patch (maximum non-irritant concentration).
Rechallenge: No
Scoring schedule: Similar to OECD 406; 24 h after removal of the patch
Removal of the test substance: After 24 h


Challenge controls:
Yes, n=4
Positive control substance(s):
yes
Remarks:
(no details, but positive results)
Statistics:
No applicable
Positive control results:
Yes, but no details
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2% in physiological saline
No. with + reactions:
9
Total no. in group:
9
Clinical observations:
mean erythema score was 1.7
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2% in physiological saline. No with. + reactions: 9.0. Total no. in groups: 9.0. Clinical observations: mean erythema score was 1.7.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
2% in physiological saline
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
no skin reaction was detected
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2% in physiological saline. No with. + reactions: 0.0. Total no. in groups: 4.0. Clinical observations: no skin reaction was detected.

Results of pilot studies:  

No irritant effects at 2% test substance in physiological saline.

Interpretation of results:
other: sensitising
Conclusions:
Sensitizing in the GPMT
Executive summary:

The study is comparable to OECD guideline 406 with acceptable restrictions (low number of negative controls; partly limited documentation).  

Under the condition of this GPMT (guinea pig maximisation test) sensitizing effects were recorded (9/9 animals with positive reaction) after challenge with 2% formaldehyde in physiological saline (non-irritant) in 9/10 animals induced via intradermal injection with 0.25% formaldehyde and topical induction with 10% formaldehyde in physiological saline (irritant).

Conclusion: Sensitizing in the GPMT.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Partly limited documentation, e.g. no data about clinical symptome, irritation, body weight, details on test substance; labelling with 3H-thymidine at day 4 instead of day 6 (see OECD 429)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(labelling with 3H-thymidine at day 4 instead of day 6)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Formalin, source BDH Ltd. (presumably aqueous solution of formaldehyde in 10% methanol); no further details
Species:
mouse
Strain:
CBA/Ca
Sex:
not specified
Details on test animals and environmental conditions:
- Source
Lab A: Animal Breeding Unit, Alderly Park
Lab B: Animal Breeding Unit, Environmental Safety Lab
Lab C & D: Harlan Olac Ltd., Bicester, Oxon
- Age/weight at study initiation: 8-12 weeks-old/ no data
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Vehicle control (4:1 acetone/olive oil), 5, 10, or 25% test substance
No. of animals per dose:
4 (control n=4) in each of 4 independent labs
Details on study design:
Topical exposure to the allergen (25 µL of the test substance solution applied to the dorsum of both ears, daily applications on 3 consecutive days) causes lymphocyte activation in the lymph nodes draining the site of application; proliferation of lymph node cells determined after labelling of cells with 3H-methyl thymidine (i.v. injection of 3H-thymidine on the 4th day and mice sacrificed 4 h later). Lymph nodes excised 5 h after i.v. injection of labelled thymidine and lymph nodes pooled for each dose group. Incorporation of 3H-thymidin measured via ß-scintilation counting.
Positive control substance(s):
not specified
Statistics:
no
Positive control results:
positive results with formaldehyde and other test substances in parallel experiments
Parameter:
SI
Value:
> 3
Test group / Remarks:
5%, 10%, 25% (all test groups)
Remarks on result:
other: Increase in stimulation index in all four laboratories; even at the low concentration of 5% the indices were > 3. Range of stimulation indices in all 4 labs: 3.7-11.9
Parameter:
other: disintegrations per minute (DPM)
Test group / Remarks:
5%, 10%, 25% (all test groups)
Remarks on result:
other: Significant increase in 3H-thymidine incorporation even at the lowest concentration of the test substance (details in Table below)

Concentration of the test substance

Lab A

Lab B

Lab C

Lab D

in %

Incorporation of 3H-thymidin in pooled lymph node cells measured in dpm x  10-2

Vehicle control

2.3

5.1

1.4

2.2

5

20.7*

18.7*

9.6*

10.0*

10

24.4*

20.6*

8.7*

10.2*

25

27.2*

29.3*

9.3*

9.2*

*: positive in the local lymph node assay

Significant increase in 3H-thymidine incorporation even at the lowest concentration of the test substance used for lymphocyte activation. Positive results in 4 independent laboratories.

Interpretation of results:
other: sensitising
Conclusions:
These results suggested skin-sensitizing activity of the test substance in the LLNA.
Executive summary:

The study is comparable to guideline study with acceptable restrictions (partly limited documentation, e.g. no data about clinical symptome, irritation, body weight, details on test substance; labelling with 3H-thymidine at day 4 instead of day 6).

In this Local Lymph Node Assay (LLNA) 4 mice per dose received toppical application of 25 µL of the test substance solution (vehicle, 5, 10, 25%) to the dorsum of both ears (daily applications on 3 consecutive days); at day 4 lymph nodes were excised 5 h after i.v. injection of 3H-labelled thymidine and lymph nodes pooled for each dose group. Incorporation of 3H-Thymidin measured via ß-scintilation counting.

A significant increase in 3H-thymidine incorporation even at the lowest concentration of the test substance used for lymphocyte activation was observed. The results were positive in 4 independent laboratories. The increase in stimulation index in all four laboratories was >3 even at the low concentration of 5% formaldehyde.

Conclusion: Formalin is sensitizing in the LLNA.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
partly limited documentation, e.g. no data about purity of the test substance; low number of test animals per trial, but 2 independent replicates; no clear data about concentration used for induction; no data about local effects after induction
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Study according to Buehler EV [1965] Archs Derm 91: 171ff
GLP compliance:
not specified
Type of study:
Buehler test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Buehler test has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Specific details on test material used for the study:
Formalin (37% formaldehyde), source Baker Chemicals, NJ, USA (presumably aqueous solution of formaldehyde in 10% methanol).
No further data.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
- Source: “commercial sources”
- Age/weight at study initiation: No data / ca. 350 g
- Number of animals per group: 10 (control n=10)
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
no data, assumed to be 0.5 ml of a solution of 5% formalin (37% corresponding to 1.85% formaldehyde)
Day(s)/duration:
Day 1, 7, and 14 for 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
2%, 0.5 mL
Day(s)/duration:
Day 28, duration 24h
No. of animals per dose:
10 controls and 30 treated animals (3 replicates a 10 animals)
Details on study design:
- Induction schedule
Animals clipped on the left flank and 0.5 mL test substance (no data about concentration) applied to this area and covered with Blenderm® and fixed with Elastoplast® and adhesive tape. Exposure period 6 h. Application repeated after 7 and 14 days. Occlusive (dermal)
- Concentrations used for induction
No data but assumed to be 0.5 ml of a solution of 5% formalin (37% corresponding to 1.85% formaldehyde). According to guideline concentration should cause mild irritation.

- Challenge schedule
One challenge on day 28; 0.5 mL on the right flank, same procedure than that used for induction but exposure period 24 h (6 h recommended).
Concentrations used for challenge: 2% formaldehyde in physiological saline, 24 h occlusive patch
(should be maximum non-irritant concentration; this concentration was also used in a Buehler assay by Hilton et al. 1996, see Section 7.5.1). No rechallenge. Removal of the test substance after 24 h.

- Scoring schedule: No data
- Positive control substance No data
- Pilot study: no data
Challenge controls:
No skin reaction was detected
Positive control substance(s):
not specified
Statistics:
No data
Positive control results:
No data
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
0
Total no. in group:
30
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
0
Total no. in group:
30
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10

0/30 animals with positive reaction. In control animals (without induction) no skin reaction was detected.

In parallel experiments the Guinea pig maximization test was conducted (guinea pigs, n= 10; control n=10, questionable concentration of formaldehyde for induction, 2% for challenge, 3 independent replicates; positive response rate 20%).

Interpretation of results:
other: ambiguous
Conclusions:
Formaldehyde (formalin) was not sensitizing in the Buehler assay but success of induction was questionable.
Executive summary:

Comparable to guideline study with acceptable restrictions (partly limited documentation, e.g. no data about purity of the test substance; low number of test animals per trial, but 3 independent replicates; no clear data about concentration used for induction; no data about local effects after induction).

10 guinea pigs per trial (3 replicates) were induced three times at day 1, 7, and 14 with formaldehyde solution of 2% (no data about local effects). For challenge at day 28 2% formaldehyde solution was used; no skin reaction was detected in 30 animals.

Conclusion: Formaldehyde (formalin) was not sensitizing in the Buehler assay but success of induction was questionable.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Sensitisation in experimental animals

Guinea pig maximisation test (GPMT):

In a Guideline study (Hoechst AG, 1983) with acceptable restrictions (no data on positive controls [but clearly positive results]; no data on impurities of the test substance [but analytical grade]; high concentration for intradermal induction) using the GPMT 20 female guinea pigs were induced by intradermal injection and topical application with 5% formaldehyde, 10 female controls were sham treated. Challenge was performed with 2 or 4% solution in test animals and controls; controls showed no erythema at both concentrations. In test animals, 4% formaldehyde resulted in a positive reaction in all tested animals and 2% formaldehyde gave 80% positive reactions at the 1st reading.

Using a similar experimental designs further studies on the GPMT (Bayer AG, 1985 & 1987) gave also positive results, even after a 2nd challenge with 0.5% formaldehyde (Bayer AG, 1985).

Kimber et al. (1991) found a positive reaction after challenge with 2% formaldehyde in physiological saline (non-irritant) in 9/10 animals induced via intradermal injection with 0.25% formaldehyde and topical challenge with 10% formaldehyde in physiological saline (irritant). Sensitising effects in all 10 treated animals were reported by Hilton et al. (1996) using the same experimental design.

In conclusion: Clearly sensitizing effects were found in the GPMT.

Buehler test:

In the study of Marzulli et al. (1982) 10 guinea pigs per trial (3 replicates in total) were induced three times at day 1, 7, and 14 with 0.5 ml of a solution of 5% formalin (37% corresponding to 1.85% formaldehyde under occlusive dressing (no data about local effects). For challenge at day 28 using the same exposure conditions; no skin reaction was detected in 30 animals. Conclusion: Formaldehyde (formalin) was not sensitising in this Buehler assay. The authors concluded that this procedure will only identify strong or moderate human contact sensitizers, but not weak ones. However, clearly positive results were presented by Hilton et al. (1996). Ten guinea pigs were induced by topical application of 5% formaldehyde solution for 6 h under occlusive dressing; this procedure was repeated once weekly for 3 consecutive weeks. Five controls were sham treated. The challenge (6 h occlusive patch with 1% test substance) was performed 12 -14 days after the last induction. A positive reaction was found in 7/10 animals. No positive reaction was detected in 5 negative controls.

Conclusion: Formaldehyde was sensitizing in the Buehler test if sufficient concentrations were used.

Local Lymph Node Assay (LLNA):

Kimber et al. (1991) conducted an interlaboratory comparison using the LLNA (comparable to guideline study with acceptable restrictions). In this LLNA 4 mice per dose received topical application of 25 μL of the test substance solutions (vehicle only, and 5, 10, 25%) to the dorsum of both ears (daily applications on 3 consecutive days); at day 4 lymph nodes were excised 5 h after i.v. injection of 3H labelled thymidine and lymph nodes pooled within each dose group. Incorporation of 3H-Thymidin was measured via ß-scintillation counting. A significant increase in 3H-thymidine incorporation even at the lowest concentration of the test substance used for lymphocyte activation was observed. The results with formaldehyde were positive in 4 independent laboratories. The increase in stimulation index in all four laboratories was >3 even at the low concentration of 5% formaldehyde.

In the LLNA of Basketter et al. (2001) 4 mice per dose received 25 μL of the test substance solution at various concentrations (0, 0.04, 0.2, 0.4, 1.9, 3.7% formaldehyde); test solutions were prepared by dissolving formalin (37% formaldehyde) in acetone/olive oil (4:1); authors used a similar experimental design as in the study above. The stimulation index increased dose-dependently. No induction was detected at 0.04% formaldehyde and the lowest sensitizing dose was 0.2% with a stimulation of proliferation by a factor of 1.9.The EC3value (3-fold stimulation of proliferation as an index of the relative potency of a contact allergen) was 0.35% formaldehyde corresponding to 87 μg/cm² (EC3 [%] x 250 [μg/cm²/% ] = EC3 [μg/cm²]). Correspondingly, the NOAEC of 0.04% formaldehyde would be 10 µg/cm². This calculation was carried out according to ECHA (2008, Chapter R.8) assuming a dose volume of 25 µl (according to the standard LLNA protocol) and an estimated application area of 1 cm² for the mouse ear. Hilton et al. (1998) reported slightly higher EC3 values (but still in the same range) using formaldehyde solutions in acetone and dimethylformamide: 0.18 mol/l (corresponding to 0.54% in acetone) and 0.11 mol/L (corresponding to 0.33% in dimethylformamide).

For risk assessment a decision has to be made which of the EC3 values obtained with different solvents should be used. For formaldehyde being very hydrophilic a highly polar solvent is most appropriate like acetone. There is no justification for the addition for a solvent containing 25% olive oil corresponding to the acetone/olive oil system of Basketter et al. (2001). Application in dimethylformamide, being a potent enhancer of skin penetration, would not correspond to exposure conditions in praxi (generally aqueous solutions). Therefore the EC3 obtained with acetone as solvent is preferred for risk assessment. The EC3 of 0.54% corresponds to an area dose of 135 µg/cm² as calculated according to ECHA (2008), which was also used by Griem et al. (2003). The authors compared quantitative data from humans and animals on skin sensitization for a large number of chemicals, including formaldehyde, and proposed a risk assessment methodology based on EC3 values from the LLNA. This value of 135 µg/cm² should be taken as the LOAEL for risk assessment based on the EC3. Correspondingly, the NOAEC for an acetone solution would be 0.06% with an area dose of 15 µg/cm². A clearly higher EC3 of 0.96% (corresponding to 240 µg/cm²) was reported by De Jong et al. (2007) using the acetone/olive oils system (see below). In addition, in order to enhance possible low responses of weak sensitizers, animals were pre-treated on the dorsum of the ears with sodium dodecylsulfate 1 h before administration of the test solutions. Thus, taking the Hilton et al. (1998) data in acetone for risk assessment represents a conservative approach. 

De Jong et al. (2007) investigated the effect of prolonged repeated exposure to formaldehyde on the magnitude of response elicited in the LLNA with a different mouse strain. After a single exposure regime according to the standard (short-term) LLNA procedure (treatment on days 0, 1, and 2 and measuring the response on day 5) the EC3 value was 0.96 % and the EC2 value 0.6 % formaldehyde. After prolonged repeated exposure (treatment on day 0, 1, and 2 followed by a treatment once a week over 7 weeks and a 3 day treatment at days 56, 57, and 58 and measuring the response on day 61) the increase of cell proliferation at 0.6 % formaldehyde was compared to that obtained by the standard (short-term) procedure. The short-term procedure led to an increase of proliferation (compared to the vehicle control group) by a factor of 2.2 and the repeated prolonged exposure by a factor of 7 with 0.6% formaldehyde. Thus, repetitive and prolonged exposure (in total 13 times over 8 weeks) increased the response by a factor of approximately 3 in comparison to short-term exposure. Obviously the response to repeated exposures is leading to a plateau as 7 repetitions of the standard (“acute”) LLNA only led to an enhancement of effect by a factor of 3.

According to the authors the finding for formaldehyde (and some other formaldehyde releasers) is in contrast to observations with some other skin sensitizers like benzocaine, dinitrochlorobenzene, or tetramethylthiuramdisulfide. These latter chemicals did not induce an increase of proliferative response by prolonged exposure under comparable experimental conditions. The authors postulate that the increased proliferated response of formaldehyde after repeated prolonged exposure may be explained by local crosslinking of proteins and other macromolecules in the skin leading to prolonged presence in the tissue. 

If these data are taken into account for risk assessment the threshold obtained by the standard LLNA procedure should be divided by 3 for extrapolation to repeated prolonged exposure. This factor should be sufficient as repeated exposure obviously lead to a “plateauing” of the magnitude of response. The NOAEC of 0.04 % (Basketter et al., 2001) for the acetone/olive oil preparation would thereby be reduced to 0.013 % corresponding to 3.3 µg/cm². Transformation of this NOAEC to the acetone solution of Hilton et al. (1998) would give a NOAEC of 0.02% (5 µg/cm²). The EC3 in acetone of Hilton et al. (1998) would be reduced to 0.18% corresponding to an area dose of 45 µg/cm².

These data suggested that formaldehyde is a strong sensitizer (cf ECHA, 2008; Table R. 8-23, p. 127).

Conclusion: Formaldehyde is sensitizing in the LLNA (with EC3 values ranging between 0.33% and 0.96% depending on the solvent and the strains used, and a NOAEC in one experiment of 0.04%).

Overall, the animal data show that formaldehyde is a skin sensitizer.

Sensitisation humans information

Formaldehyde is a dermal allergen in humans (WHO, 1989; IARC, 1995; ATSDR, 1999; OECD, 2004; summary of sensitisation in humans). Formaldehyde solution is a primary skin sensitizer inducing allergic contact dermatitis Type IV and may induce contact urticaria Type I (WHO, 1989). However, contact urticaria has been rarely associated with formaldehyde exposure (IARC, 1995; MAK, 2010). In the OECD SIDS Formaldehyde (2004; see summary of sensitisation in humans) a threshold for the challenge concentration in patch tests on formaldehyde sensitized subjects was reported: 30 ppm (0.003%) in aqueous solution and 60 ppm (0.006%) for products containing formaldehyde (but no reference is given by OECD in this respect). However, other data on concentration-response relationships for skin allergic reaction in formaldehyde-sensitive patients induced by dermal exposures to formaldehyde suggested that a positive reaction to formaldehyde is rare below concentrations of 0.025-0.05% (ATSDR, 1999).

An indication for a threshold for induction of allergic skin reactions in sensitized persons can also be obtained from formaldehyde concentrations used for diagnostic patch testing. Trattner et al. (1998) compared the results obtained by simultaneous testing with 1% and 2% formaldehyde solutions in 3734 patch tests. Testing was done by occlusion in Finn Chambers and the patches were applied to the skin for 2 days. The number of positive reactions was the same for both concentrations, but 2% gave significantly more irritant reactions and therefore a 1% patch test concentration was proposed. But this concentration is a compromise between avoiding irritation (false positive) and sufficient sensitivity for detection of sensitization. As indicated in the review of De Groot et al. (2009) even an 1% test concentration may lead to false positive reactions in about 50% and as the study of Trattner et al. (1998) indicates about 40% of allergic patients may be missed in comparison to those identified by a test concentration of 2%.

Several studies have investigated the threshold for elicitation of contact allergic reactions in patients allergic to formaldehyde. Jordan et al. (1979) performed double-blind controlled tests on formaldehyde threshold responses in 9 allergic patient by repeated closed patch testing at the same site for 1 week (three patches applied during the week) with 0, 30, 60, and 100 ppm formaldehyde. Five patients were known to have a strong allergy to formaldehyde. The following incidences of positive reactions were found: 4/9 at 30 ppm; 5/9 at 60 ppm; 6/9 at 100 ppm. Two subjects reacting to 30 ppm were retested later and again had positive reactions. As the closed patch test represents exaggerated exposure conditions in comparison to many exposure scenarios, 11 subjects with delayed hypersensitivity to formaldehyde (including 7 from the closed patch testing) were tested in an open approach by spraying 30 ppm formaldehyde in an axilla (twice a day over 2 weeks). Only a very minimal response was produced in 2/11 subjects. The authors conclude that levels below 30 ppm should be tolerated by sensitive subjects if repeatedly applied to normal skin. It should be mentioned that an exaggerated closed test procedure with 3 applications over 1 week was used here in comparison to the standard closed patch testing once over 48 h. Finally, the description of the test method (no data for area of application and total amount administered) does not allow a calculation of the amount of formaldehyde applied per unit area.

Flyvholm et al. (1979) tested 20 patients allergic to formaldehyde with serial dilutions of 25, 50, 250, 500, 5000, and 10000 ppm formaldehyde under occluded conditions. 15 µl formaldehyde solutions were applied for 2 days by Finn Chambers (diameter 0.8 cm corresponding to an area of 0.5 cm²). Nineteen reacted to 10000 ppm, 9/20 to 5000 ppm, 2/20 to 500 ppm and 1/20 to 250 ppm. Retesting of the patient reacting to 250 ppm 1 year later showed negative results with 50, 100, and 250 ppm. In addition, non-occluded patch testing was performed by application of 15 µl of the formaldehyde solutions to a 1 cm² area of the forearm and allowing to dry at room temperature. Under these conditions no positive reactions were observed even at the highest concentration of 10000 ppm. The authors concluded that 250 ppm formaldehyde is the threshold concentration for occluded patch testing. The Finn Chamber device (area 0.5 cm²) used here allows the calculation of the amount of formaldehyde per unit area: 7.5 µg/cm² for 15 µl with a concentration of 250 ppm. Griem et al. (2003) assumed a NOAEC of 100 ppm corresponding to an area dose of 3µg/cm².

Finally, De Groot et al. (1988 cited in De Groot, 2009) found that 8 out of 35 formaldehyde allergic patients reacted with closed patch testing down to 1000 ppm, lower concentrations were not investigated.

Based on these findings De Groot et al. (2009) concluded that the safe formaldehyde concentration for sensitive patients remains largely unknown. Levels of 200-300 ppm free formaldehyde in cosmetic products have been shown to induce dermatitis from short-term use on normal skin.

In conclusion, by these data it is proposed to take 3 µg/cm² as NOAEL in formaldehyde sensitized persons for risk assessment.

A threshold concentration for induction of formaldehyde sensitization has been estimated to be less than 5% aqueous solution (OECD, 2004). This probably relates to formalin (and not formaldehyde) as used in the study of Kligman (1966, cited in OECD, 2004). The author described a human Maximization Test with formalin. For induction 5% formalin (formaldehyde concentration not given) was applied under occlusive dressing 5 times over 48 h preceded each by occlusive application of sodium lauryl sulfate over 24 h to induce moderate inflammation. The challenge test used 1% formalin after pretreatment with sodium lauryl sulfate for 1 h. Thereby 18/25 subjects became sensitized. As no further concentrations were tested, a threshold for induction of skin sensitoization cannot be derived from this publication.

Marzulli and Maibach (1974) used the Draize test to investigate the induction concentration of formaldehyde in human volunteers by the human repeated-insult test (HRIPT). For induction the material was applied to the skin under occlusive conditions for 48 or 72 h. Ten epicutaneous applications were administered at the same site within 3.5 weeks. After a rest period of 2 weeks a challenge patch containing 0.37% formaldehyde was applied for 72 h. The following results were obtained for different induction concentrations of formaldehyde: 0.037%: 0/45; 0.37%: 4/89; 1.1%: 5/88; 1.9%: 4/52; 3.7%: 8/102 (subjects with positive reactions / subjects tested). Thus, induction of sensitization is not to be expected at a concentration of 0.037% even after repeated exposures under occlusive dressing over 2-3 days. The NOAEC of 0.037% was transformed to an area dose of 37 µg/cm² in reviews by Griem et al. (2003), Gerberick et al. (2001) and Basketter et al. (2005, 2008). In their review Basketter et al. (2008) note some shortcomings of the methodological description by Marzulli and Maibach (1974) but by taking account of the exposure being greater than in the standard HRIPT protocol they concluded that 37 µg/cm² is a reasonable conservative estimate of the NOAEC.

In a recent study (Thompson et al., 2002) patch testing in patients with suspected allergic contact dermatitis was performed in thebetween 1994 and 1996 (standard tray of 49 allergens including formaldehyde). Patients were exposed to 1% formaldehyde in water for 48 h and readings were performed 48 h after patch removal. In Kansas City 20 out of 138 patients with dermatitis showed a positive result in the patch test (corresponding to 14.5%); in other regions of the USA 268 out of 2973 patients patch tested with formaldehyde showed a positive result (corresponding to 9.0%). The difference is significant (p<0.05). Further details on the dermal sensitization rate for patients tested in dermatological clinics are given by MAK (2010). Inthe prevalence has nowadays declined to about 2%. Fora rate of 0.9% was reported for the general population and inof 0.3-0.6%.

Conclusion: In the USA 9% of patients with allergic contact dermatitis showed positive reaction in the standard patch test with 1% formaldehyde, in Kansas City 20 out of 138 patients (14.5%). Lower incidences have been described for.

With regard to skin sensitization MAK (2010) concluded that allergic contact dermatitis is relatively frequently observed in patients by diagnostic patch testing. In addition, many experimental animal studies gave positive results for sin sensitization.

A literature search after the last IUCLID update was carried out up to April 20, 2015 generally reinforcing the skin sensitising potential of formaldehyde.

Zug et al. (2009, supporting) reported results from 4454 patients tested for 65 allergens by the North American Contact Dermatitis Group in 2005-2006. Formaldehyde ranged among the 15 most frequent allergens with an incidence rate of 9% (for comparison, the incidence rate for nickel sulphate was 19%, the highest incidence observed). Similar results were obtained by Arrandale et al. (2012, supporting): formaldehyde ranged among the 10 most frequent skin allergens. In 3676 patients 19 reacted positive to formaldehyde and the response was related to workplace exposure. When 6845 patients in Australia were patch tested between 1993 and 2006, formaldehyde was the most frequent contact allergen among the preservatives studied with 4.6% positive results (Chow et al., 2012, supporting).

Lundov et al. (2010, supporting) found by diagnostic patch testing that patients allergic to formaldehyde-releasers often had simultaneous allergy to formaldehyde itself. Nearly all patients who reacted positively to 2 formaldehyde-releasers were also allergic to formaldehyde. Thus concomitant allergy to formaldehyde-releasers and formaldehyde is common.

Isaksson et al. (2011, supporting) found that in 2504 patients that were tested by standard patch test series, 1.1% (27 subjects) reacted positively to a mixture of monomers and dimers from a resol resin based on phenol and formaldehyde. Of these 27 patients 2 reacted positively in addition to formaldehyde and 2 to a p-tert-butylphenol-formaldehyde resin. The authors propose that the mixture they used here should be added to the standard battery of substances for diagnostic patch testing.

Ponten et al. (2012, supporting) found that increasing the concentration of formaldehyde for diagnostic patch testsing from 1% (as commonly used in clinical practice) to 2% (wt/vol) significantly increased the positive reactions from 1.8% to 3.4%. Therefore, they propose that 2% test solutions should be used for clinical diagnostic patch testing.  

Kadivar and Belsito (2015, supporting) patch tested 2611 patients, among these 165 health care workers. Health care workers had significantly more workplace related allergic contact dermatitis, especially to formaldehyde, than non-health care workers.

Systemic sensitization (immediate type of allergy mediated by IgE)

An anaphylactic shock reaction after accidental i.v. application of formaldehyde during hemodialysis due to formaldehyde remaining in the system after disinfection with formaldehyde has been described in a case report (WHO, 1989). However, no data were given on the amount of formaldehyde applied. Furthermore, this type of sensitization seems to be very rare.

In the study of Kunisada et al. (2002;) anaphylaxis due to formaldehyde released from root-canal disinfectant has been reported. The results of this study suggested anaphylaxis due to Type-I alIergy to formaldehyde.

Classification and labelling of active substance according to Directive 1999/45/EC: R43 IARC (2006) presented a summary on toxic effects in animals (see Section Other effects) as well as a summary on toxic effects in humans including data on sensitisation.

For risk assessment it is proposed to take an area dose of 3 µg/cm² in subjects allergic to formaldehyde and of 37 µg/cm² for induction of formaldehyde contact dermatitis based on human data. Based on animal data (LLNA test with acetone) an area dose of 135 µg/cm² for the EC3 and of 15 µg/cm² for the NOAEC for induction is proposed. Division by 3 to take into account repeated exposure would give an EC3 of 45 µg/cm² and a NOAEC of 5 µg/cm².


Respiratory sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Limitations in design and/or reporting, but adequate for assessment. No appropriate positive control substances that may lead to dermal sensitisation with or without being respiratory sensitisers.
Principles of method if other than guideline:
The role of air humidity and allergic sensitization on the acute airway response to inhaled formaldehyde (FA) vapor was investigated in mice. Mice were sensitized to the immunogen ovalbumin (OVA) by three intraperitoneal injections followed by two aerosol challenges. Once sensitized, the mice were housed at high (85–89%) or low (<10%) relative humidity, respectively for 48 h prior to a 60-min exposure to either 0.4, 1.8 or about 5 ppm FA. Before, during and after exposure, breathing parameters were monitored.
GLP compliance:
not specified
Specific details on test material used for the study:
Name of test material (as cited in study report): Formaldehyde
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic, Denmark
- Housing: Polypropylene cages (380 × 220 × 150 mm) with pinewood sawdust bedding (Lignocel S8, Brogaarden, Denmark).
- Diet: Altromin no. 1324, Altromin, Lage, Germany, ad libitum
- Water:ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30–50
- Photoperiod (hrs dark / hrs light): 12 / 12 (6 a.m. to 6 p.m.)
Concentration:
- Target concentrations were 0.4, 1.8 and 7 ppm,
- The concentrations measured in the animal exposure chamber were similar under dry and humid environments both at the low (0.42 ± 0.01 ppm) and medium (1.8 ± 0.09 ppm) FA levels. At the highest FA level, the concentrations were 4.0 ppm and 5.7 ppm in the dry and humid environments, respectively.
No. of animals per dose:
30
Details on study design:
Sensitization:
- Mice (n = 30) were immunized to OVA by intraperitoneal (i.p.) injections of 1 μg OVA in combination with 270 μg Al(OH)3 in 100 μL 0.9% saline on day 0.
- Mice were boosted i.p. on days 14 and 21 with 0.1 μg OVA in 100 μL 0.9% saline.
- Finally, the animals were exposed 20 min to an aerosol of 0.2% OVA on days 28 and 29 using a Pari Star nebulizer (PARI GmbH, Starnberg, Germany),
- For the non-sensitized control mice (n = 30), saline (0.9%) was used for the three i.p. injections and the aerosol exposures.
- On days 29 and 30, the sensitized and non-sensitized mice were housed at either low (b10) or high (85–89) % relative humidity.

Formaldehyde exposure and exposure conditions:
- On day 31, the mice were exposed to FA.
- Formaldehyde was generated from a Kin-Tek (TX, USA) gas standard generator (Model 491MB) by use of a permeation tube and dry air, and led to a 24 L exposure chamber.
- The airflow rates in the chamber were set at 18.8 - 23.2 L/min.
- The chamber exposure concentrations of FA were monitored pre- and post-FA exposures and every 10 min during exposures by 10 min air sampling of 4.4 L on dinitrophenylhydrazine (DNPH) sampling cartridges.
- Samples were alyzed by HPLC using a diode array detector.
- Mice were inserted into body plethysmographs that were connected to the exposure chamber. The respiratory parameters were obtained for each mouse from a pneumotachograph connected to each head-out plethysmograph that allows continuously monitoring of the parameters.
- The exposures were preceded by a period that allowed the mice to adapt to the plethysmographs. Then, a 15 min period was used to establish baseline (control) values of the respiratory parameters. This period was followed by a 60 min exposure period and a 30 min recovery period.
Challenge controls:
Non-immunised animals
Results:
Lung inflammation:
- No effect was seen on FA exposure and humidity on the degree of lung inflammation. Only the sensitization procedure increased the number of BAL cells.

Airway effects of FA in sensitized mice:
- Humid environment:
-- In the humid environment, little effect of allergic sensitization was seen in the upper respiratory tract, as suggested by the superimposed Time of Break (TB) responses. As typical for nose irritation, the maximum response was reached within the first 20 min of exposure period and thereafter gradually reduced during the remaining part of the period. After cessation of exposure (at time = 60 min), the TB response rapidly approached the baseline level. No apparent difference was revealed by comparison of the AUCs from the OVA and saline sensitized mice, respectively. Similar results were seen at the two lowest FA concentrations.
-- Sensitization to OVA amplified the effect of FA in the conducting airways at the highest FA concentration but not at lower concentrations.
-- The stronger response from the lungs at the higher FA concentrations was also to some extent reflected by a marginally elongated Time of Pause.
-Dry environment.
-- Saline control mice had a high, short-lasting peak in TB, suggesting a vigorous, transient nose irritation
-- OVA-sensitization reduced the airway responsiveness to FA in the conducting airways compared to the saline control mice. Thus, both at the medium and high FA exposure concentrations, the saline sensitized control mice showed a more pronounced reduction in VD compared to the OVA-sensitized mice.

Effect of relative air humidity on airway response to FA
- Saline control mice: No effect of humidity was apparent on nasal irritation in the non-sensitized mice even at high concentrations of FA
- The humidity had an impact on the conducting airways at FA >1.8 ppm. Hence, mice housed in a dry environment had a more pronounced decrease in expiratory flow at the highest FA concentration compared to mice housed in the humid environment. The amplifying effect of a dry environment on lower airway effects was also apparent from the TP; it showed that the highest FA concentration induced a stronger pulmonary irritation in the dry environment compared to the humid environment.

Conclusion: Formaldehyde induced airway irritation was both influenced by air humidity and prior allergic airway inflammation, but only at high concentrations of ≥1.8 ppm. There was no interaction at 0.4 ppm, a concentration relevant for occupational and indoor exposures.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Limitations in design and/or reporting, but adequate for assessment. No known pulmonary sensitisers as positive controls substance included in study design.
Principles of method if other than guideline:
Balb/c mice were randomly divided into six groups (6/group): (1) saline control; (2) ovalbumin (OVA)-immunized (OVAimm) only; (3) 0.5 mg FA/m3 exposure; (4) OVAimm + 0.5 mg FA/m3; (5) 3.0 mg FA/m3 FA exposure; and, (6) OVAimm + 3.0 mg FA/m3. Experiments were conducted after 3 week of combined exposure and a 1-week challenge with aerosolized OVA. Airway hyper-responsiveness, pulmonary tissue damage, eosinophil infiltration, and increased interleukin (IL)-4 and IL-6 levels and interferon levels in lung tissues were measured.
GLP compliance:
not specified
Specific details on test material used for the study:
Name of test material (as cited in study report): Formaldehyde (FA)
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hubei Experimental Animal Center (Wuhan, China)
- Age at study initiation: 7–8-week-old
- Weight at study initiation: 18–20 g
- Housing: pathogen-free room

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–25
- Humidity (%): 50–70
- Photoperiod (hrs dark / hrs light): 12/12
Route of induction exposure:
inhalation
Remarks:
Whole body
Route of challenge exposure:
inhalation
Vehicle:
other: air
Concentration:
0.5 and 3.0 mg/m³
No. of animals per dose:
2x 6 animals
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- Exposure period: 6 h/day
- Test groups: 6 groups
(1) saline control: No formaldehyde exposure, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution.
(2) Ovalbumin (OVA)-immunized (OVAimm) only: No formaldehyde exposure, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution containing 20 mg OVA (Sigma) and 20 mg aluminum hydroxide each time.
(3) 0.5 mg/m3 exposure: Exposure to 0.5 mg/m³ formaldehyde, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution
(4) OVAimm + 0.5 mg/m3: Exposure to 0.5 mg/m³ formaldehyde, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution containing 20 mg OVA (Sigma) and 20 mg aluminum hydroxide each time
(5) 3.0 mg FA/m3 FA exposure: Exposure to 3 mg/m³ formaldehyde, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution.
(6) OVAimm + 3.0 mg/m3: Exposure to 3 mg/m³ formaldehyde, each mouse was injected intravenously on Days 10 and 18 with 200 μl of a normal saline solution containing 20 mg OVA (Sigma) and 20 mg aluminum hydroxide each time
- Site: inhalation
- Frequency of applications: Formaldehyde exposure daily
- Duration: Formaldehyde exposure 21 days
- Concentrations: 0.5 mg/m³ and 3.0 mg/m³

B. CHALLENGE EXPOSURE
-Test groups: 6 groups
(1) saline control; aerosol challenge with saline for 30 min/d on Days 22–28 using an ultrasonic nebulizer.
(2) ovalbumin (OVA)-immunized (OVAimm) only; aerosol challenge with 1% OVA for 30 min/d on Days 22–28 using an ultrasonic nebulizer.
(3) 0.5 mg FA/m³ exposure: aerosol challenge with saline for 30 min/d on Days 22–28 using an ultrasonic nebulizer.
(4) OVAimm + 0.5 mg FA/m3; an aerosol challenge with 1% OVA for 30 min/d on Days 22–28 using an ultrasonic nebulizer.
(5) 3.0 mg/m³ FA exposure; an aerosol challenge with saline for 30 min/d on Days 22–28 using an ultrasonic nebulizer.
(6) OVAimm + 3.0 mg FA/m3: an aerosol challenge with 1% OVA for 30 min/d on Days 22–28 using an ultrasonic nebulizer.

To prevent adversely affecting measures of lung cytokines/pulmonary architecture due to any possible influence from the methacholine used in airway hyperresponsiveness assessments, dedicated groups of mice (for each of the above regimens) were employed.
- One set of 36 mice was treated for 28 days according to the above-regimen(s) and then used for AHR tests and BALF sample collection;
- A second set of 36 mice was treated as above and then used directly (i.e.,non-lavaged) for cytokine (left lung) and histopathologic (right lung) analyses.

Airway hyper-responsiveness as an indicator of lung function was tested 24 h after the final aerosol exposure using an AniRes 2005 lung function system. When the baseline parameters were stable, a 27-gauge needle was inserted into the jugular vein and methacholine (O-acetyl-β-methylcholine chloride,MCh;) was administered through a catheter; increasing dosages of 0, 0.025, 0.05, 0.1, and 0.2 mg MCh/kg were injected every 5 min. Mouse airway responsiveness in each dosing cycle was examined by measuring expiratory resistance (Re), inspiratory resistance (Ri), and dynamic lung compliance (CLdyn).

BALF was collected immediately after the conclusion of the airway hyper-responsiveness measurements. Total cell counts were obtained from 500 μl of the cell suspension using a blood cell analysis system. Another 500 μl was prepared for eosinophil counts. Eosinophils were stained using Eosin Y sodium solution, and then assessed using a hemacytometer and a DM 4000B microscope

Pulmonary histopathological analyses: Right lungs from non-lavaged mice were isolated for histopathology slice preparation. Results were then reported in terms of
- slight: a slight thickening of the airway wall without damage/folding on the cuticular layer of the airway, or cell infiltration around airway and vessels
- moderate: a bit of thickening of the airway wall and there some folding on the cuticular layer, and a little cell infiltration around airway and vessels
- severe changes. obvious thickening of the airway wall and severe damage/folding on the cuticular layer, with mucus insidethe airway and great amounts of cell infiltration around airways and vessels.

Detection of the IL-4, IL-6, and interferon (IFN)-γ: Levels of IL-4, IL-6, and IFNγ in non-lavaged left lung tissue homogenates were assessed using commercial ELISA kits according to manufacturer’s instructions.
Results:
The authors conclude that formaldehyde can induce and aggravate asthma in mice.

Assessment of airway hyper-responsiveness:

- All of the OVA-treated mice (group 2, 4, 6) exhibited greater airway responses to MCh when compared with those of the saline controls.

- The FA 0.5 + OVA and FA 3.0 + OVA groups had a significantly enhanced airway response compared with the OVA-immunized only group and the saline control group.

- The FA 3.0 + OVA mice showed a very marked change in their airway responses compared with mice in the OVAimmunized- only group, resulting in an extreme decline in lung function.

- The two groups exposed to FA only (FA 0.5 and FA 3.0) did not display any significant changes (relative to controls), even though their responsiveness was higher than that of the saline mice but lower than that of the OVA-immunized-only mice.

Pulmonary histopathology:

- All OVA-treated groups showed clear pulmonary histopathological including inflammatory cell infiltration, airway wall thickening, and remodeling.

- In FA 3.0 + OVA mice the most severe changes, including cell infiltration and airway remodeling, were found.

- in the FA 3.0 group slight changes were observed compared with the saline control group.

Eosinophil infiltration in BALF:

- In all formaldehyde exposed groups the ratios of eosinophils to total cells contained in BALF were all significantly increased compared with ratios among the saline controls.

- OVA immunization significantly intensified the increased eosinophil infiltration induced by FA. (p<0.01)

Cytokine detection:

- Both FA exposure and OVA-combined FA exposure enhanced IL-4 and IL-6 levels in pulmonary tissue homogenates.

- FA 3.0 exposure, but not FA 0.5 exposure, induced a significant increase in IL-4 and IL-6 levels, even compared with the OVA-immunized only group.

- FA 3.0 + OVA exposure induced the highest IL-4 and IL-6 levels.

- IFNγ levels were not significantly different among the groups.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Respiratory tract sensitization in experimental animals

Test methods to reliably differentiate between skin and respiratory sensitizers and to identify the latter are still under development. MAK (2010) has summarized the results obtained for formaldehyde. Notwithstanding the still exploratory status of these protocols, for formaldehyde no evidence for specific induction of airway sensitization can be derived from the animal studies. Basically 3 approaches have been investigated: changes in minute volume, determination of IgE antibodies, and determination of specific cytokine profiles.

Lee et al. (1984) exposed guinea pigs by the inhalation, dermal, and injection (into footpads with Freund’s complete adjuvant) routes. Pulmonary hypersensitivity was assessed by measuring a potential increase in respiration rate. No pulmonary hypersensitivity was detected by challenge with 2 or 4 ppm formaldehyde for any of the induction routes and formaldehyde-serum albumin adducts were not observed after inhalation treatment. On the other hand, dermal sensitization was observed by all of the different induction routes. Formaldehyde led to skin sensitization in guinea pigs without causing respiratory hypersensitivity.

Arts et al. (2008) developed a respiratory local lymph node assay. Mice were exposed to the test chemical by repeated inhalation and 3 days after the last exposure cell proliferation was determined in the draining mandibular lymph nodes. While several respiratory sensitizers led to an increased proliferation rate, formaldehyde did not.

Another approach was to study IgE antibody induction after dermal exposure of mice. While some typical respiratory sensitizers led to a dose dependent induction of total IgE antibody content, this was not observed after dermal treatment with formaldehyde. The animals received a total dose of up to 6.8 mg formaldehyde on the shaved flank and dorsum of the ear. The validity of this protocol was demonstrated as several diisocyanates and trimellitic anhydride (TMA) gave clearly positive results while only slight effects were found with dinitroclorobenzene and glutaric dialdehyde (Potter and Wederbrand, 1995). Similar results were obtained by Hilton et al. (1996). Mice (n = 6 per group) were exposed twice at day 1 and 7 via the skin to irritant concentration of formaldehyde solutions for induction. At day 14 the animals were killed and serum IgE concentration (indicator for respiratory allergy) was measured. In contrast to the positive control TMA (trimellitic anhydride), a known human respiratory allergen, the IgE concentrations in mice exposed to formaldehyde solutions or to the contact allergen DNCB (2,4-dinitrochlorobenzene) were not increased. It is concluded that formaldehyde lacks a significant potential to cause sensitization of the respiratory tract. Similarly, in rats topical application of formaldehyde did not lead to an increase in serum IgE concentration in contrast to the effects observed after trimellitic anhydride treatment (Arts et al., 1997). But according to the authors further studies may be needed with chemicals that have both irritant and sensitizing properties at about similar concentrations or may act through non-IgE-mediated immune mechanisms.

Several authors investigated cytokine profiles as an indicator for respiratory sensitization. In such a study (Hilton et al., 1996; no vehicle control data given) 50 μL of the test solution (10, 25, 50% formalin in DMF vehicle or 25% trimellitic anhydride [positive control] in vehicle) were applied on both shaved flanks of mice (n = 10 per group) and 5 days later the treatment was repeated. Further 5 days later, 25 μL of the same test solution was applied to the dorsum of both ears, once daily for 3 consecutive days; 13 days after initiation of exposure, mice were sacrificed and draining auricular lymph nodes were excised and pooled for each group.

Single cell suspensions were prepared of lymph node cells (LNC). Concentrations of 2 different cytokines, interferon gamma and interleukin 10, in the cell culture supernatants of draining lymph node cells were determined by sandwich ELISA (enzyme-linked immunosorbent assay) method. Formalin provoked the production of interferon gamma but did not increase the interleukin 10 concentration. The reverse pattern was detected with the positive control TMA: slight increase in interferon gamma but a clear increase in interleukin 10. These data suggested that formaldehyde did not induce IgE response in mice and that no sensitization of the respiratory tract in mice is induced.

In a similar experiment (Dearman et al., 1999) mice were treated dermally with formaldehyde, 2,4-dinitrochlorobenzene and trimellitic anhydride. Production of different cytokines (interferon-gamma, interleukin-4 and-10) was determined in the local lymph nodes. A similar cytokine profile was obtained for formaldehyde and 2,4-dinitrochlorobenzene, clearly different to that of trimellitic anhydride. The authors concluded that formaldehyde does not elicit significant allergic sensitization to the respiratory tract.

Xu et al. (2002) investigated the mRNA expression of different cytokines in spleen and draining lymph nodes of mice after cutaneous exposure to formaldehyde. They suggested that the induction of IFN-gamma and IL-4 are common immunological features of contact allergens. Dearman et al. (2005) compared frequencies of intracellular cytokine (IL-4 and IFN-gamma)-positive CD4+and CD8+lymphocytes in the draining lymph nodes of mice after topical treatment. The reactions to two contact sensitizers (dinitrochlorobenzene and formaldehyde) and two respiratory sensitizers were compared and both types showed different patterns. The respiratory sensitizers led to a much higher frequency of IL-4-expressing CD4+cells as compared to the contact sensitizers. Both types led to a similar increase in IFN-gamma positive CD4+and CD8+lymphocytes. These different patterns might be used to discriminate skin and respiratory sensitizers.

After inhalation exposure of mice (according to Arts et al., 2008) to different respiratory and contact sensitizers, cytokine production (interferon-gamma, interleukin-4 and -10) was determined in the draining lymph nodes (De Jong et al., 2009). It was proposed that interleukin-4 could specifically be used to identify respiratory sensiztizers. De Jong et al. reported that formaldehyde did not produce significant production of any cytokine after inhalation or skin exposure.

Summarizing studies up to 2001 Dearman and Kimber (2001) conclude that cytokine fingerprinting provides a reliable method for the identification of chemicals causing allergic sensitization of the respiratory tract and to distinguish these from chemicals associated primarily with allergic contact dermatitis. In contrast, in a study on cytokine expression profiles during murine contact allergy Ulrich et al. (2001) cautioned that the use of selected cytokine profiles to distinguish respiratory from skin sensitizers may not be an appropriate tool. Similarly MAK (2010) concludes that at present different cytokine profiles may not clearly differentiate between both types of sensitizers.

In summary, even under consideration that generally accepted animal testing procedures for defining respiratory sensitizers are not yet available, the weight of evidence from animal experiments indicates that formaldehyde does not act as a respiratory sensitizer.

A literature search after the last IUCLID update was carried out up to April 20, 2015.

Generally these studies are difficult to interpret because no known respiratory sensitizers were included as positive reference substances.

Liu et al. (2011, key) exposed mice at 0, 0.5, and 3.0 mg/m³ over 21 days. On day 10 and 18 ovalbumin was injected intravenously for induction of sensitisation and on days 22-28 the animals were challenged with aerosolised ovalbumin. Control groups with and without formaldehyde inhalation and ovalbumin induction were included. The method followed that of Qiao et al. (2009) for rats. After the last challenge exposure lung function was measured in trachea-cannulated mice after intravenous application of O-acetyl-ß-methacholine and bronchi-alveolar lavage fluid was collected. Histopathology of the lung was performed and various cytokines in the lung were measured. At the high formaldehyde concentration airway hyper-responsiveness, pulmonary tissue damage, eosinophil infiltration and increased interleukin IL-4 and IL-6 in lung tissue were found in the ovalbumin + formaldehyde treated animals as compared to mice only immunised to ovalbumin. The authors conclude that formaldehyde can induce and aggravate asthma in mice. Although this study did not include known pulmonary sensitizers as positive controls substances, a Klimisch score 2 (key study) is assigned because the findings closely correspond to those of Qiao et al. (2009) in rats.

Wu et al. (2013, supporting) carried out a mechanistic study to identify the mechanism and receptors involved in the asthma model of Liu et al. (2011). A similar experimental protocol was used with a formaldehyde exposure scheme of 5 d/week (6 h/d) over 4 weeks, ovalbumin induction on days 10, 18, and 25, and challenge on days 29-35. During formaldehyde exposure HC-030031 (ankyrin 1 receptor antagonist) and capsazepine (vanilloid 1 receptor antagonist) were injected daily by the intraperitoneal route. Control groups without formaldehyde, ovalbumin or the receptor antagonists were included. An extended set of parameters was measured as compared to that of Liu et al. (2011) including immunohistochemistry, cytokines and neuropeptides. Regarding the combined action of formaldehyde and ovalbumin the results of Liu et al. (2011) were confirmed and the pulmonary reaction was reduced by the receptor antagonists.

Larsen et al. (2013, key) tested acute airways effects of formaldehyde in mice sensitised by 3 intraperitoneal injections of ovalbumin on day 0, 14, and 21. On day 28 and 29 the animals were exposed to an aerosol of ovalbumin (20 min) and on day 31 to formaldehyde for 1 h (0.4, 1.8, and 5 ppm). On day 29 and 30 the mice were hold under conditions of high (85-89%9 and low (<10%) relative humidity. Before, during, and after exposure breathing parameters of the upper respiratory tract and specific markers of lung and nose irritation were measured. Non-immunised animals served as controls. Formaldehyde induced airway irritation was both influenced by air humidity and prior allergic airway inflammation, but only at high concentrations of ≥1.8 ppm. There was no interaction at 0.4 ppm, a concentration relevant for occupational and indoor exposures.

In summary, these studies cannot be taken as an experimental proof that formaldehyde may lead to respiratory sensitisation. Some of these investigations are barely acceptable because they used an exposure procedure without analytical verification of formaldehyde in the exposure system. But above all, none of these studies used appropriate control substances that may lead to dermal sensitisation with or without being respiratory sensitizers.

Respiratory tract sensitization, human information:

There are only a few case reports of bronchial asthma (2 renal dialysis nurses, a plastic moulder, a printer, a worker in a phenol formaldehyde manufacturing plant, and a carpenter) indicating respiratory tract sensitization. In acute exposure challenges at exposure levels of 3 ppm formaldehyde, all above mentioned subjects showed marked changes in FEV1 or airflow rates. In a study on 230 patients occupationally exposed to formaldehyde and who had reported respiratory symptoms consistent with asthma only 12 subjects showed a decrease inPeak Expiratory Flow Rate (PEFR) > 15% after acute challenge with 2 ppm formaldehyde. However, the mechanism of sensitization in this study is uncertain (ATSDR, 1999; WHO, 1989). In contrast to these positive results no challenge-induced deficits in FEV1 or airflow rates were demonstrated in 3 other studies on formaldehyde exposed subjects with respiratory problems (ATSDR, 1999).

The formation of formaldehyde-specific IgE antibodies in groups of formaldehyde-exposed subjects has been investigated in several studies. In general, the results did not provide evidence for a formaldehyde-induced respiratory allergy. However, there is some evidence for formation of specific IgE in children exposed to low level of formaldehyde in indoor air (Wantke et al., 1996; in ATSDR, 1999).

In contrast, Doi et al. (2003) concluded that it appears unlikely, that formaldehyde is a relevant allergen in childhood asthma.In this study specific IgE against formaldehyde was measured by CAP RAST in 122 asthmatic children and 33 nonallergic children in. A questionnaire on clinical features of their asthma, their living conditions, and symptoms of mucosal irritation was performed. The personal factors were similar in the two groups. The median level of total IgE of asthmatics (755 IU/mL) was significantly higher than that of controls (108 IU/mL). However, formaldehyde-specific IgE was detected in only two asthmatic children: one exhibited 0.42 UA/mL of formaldehyde-specific IgE and the other 0.46 UA/mL. Both values are only slightly increased compared with the limit given for positive results of 0.35 UA/mL.

One of these 2 children had severe asthma and frequent complaints of mucosal irritation in places where the formaldehyde level was likely to be high. On the other hand, the other child had mild asthma and rare complaints of mucosal irritation. In contrast, it is also stated that both patients did not have severe asthma or frequent symptoms of mucosal irritation. There was no significant difference between the control group and the group of asthmatic children in prevalence of IgE to formaldehyde. These results suggest that the prevalence of IgE to formaldehyde may be rare in Japanese children and FA may not play an important role as an allergen causing asthma or as an irritant.

MAK (2010) reviewed the literature on formaldehyde and asthma and concluded that the allergological findings do not provide a consistent pattern. In inhalation challenge tests generally immediate reactions were observed, dual or late reactions were rare. A differentiation against irritation often is difficult and specific IgE antibodies, if found, mostly did not correlate with the respiratory symptoms. Overall, a relationship of respiratory symptoms with formaldehyde is only in few cases sufficiently documented. The small number of reliable findings in comparison to the broad exposure potential would not warrant to classify formaldehyde as a respiratory sensitizer according to the criteria of the MAK commission.

Recently a theory was developed how formaldehyde might possibly exacerbate asthma and induce bronchoconstriction through influencing thiol biology (Thompson and Grafström, 2008; Thompson et al., 2008). This theory is based on the fact that that formaldehyde dehydrogenase (FDH) has a dual role in formaldehyde metabolism and also partially regulates nitrosothiol homeostasis by catalyzing the reduction of the endogenous nitrosylating agent S-nitrosoglutathine (GSNO). Thereby formaldehyde may influence FDH mediated catabolism of GSNO which acts as an endogenous bronchodilator. But taking into consideration that neither animal experiments nor observations in humans lend sufficient support to an asthmagenic action of formaldehyde, this hypothesis remains speculative.

A literature search after the last IUCLID update was carried out up to April 20, 2015. In this section studies on effects to the respiratory tract not directly related to asthma will be reported here, too.

Wolkoff and Nielsen (2010, key) reviewed the studies related to effects of formaldehyde on asthmatics especially at low exposures of <100 ppb. After a detailed analysis of the conflicting results in the volunteer studies reported by Ezratty et al. (2007) and Cassett et al. (2006) they concluded that exposure of asthmatics to formaldehyde would not lead to an exacerbation of the lung function. They also reviewed case control and cross sectional studies that had suggested a possible association of low formaldehyde concentrations and asthma. By taking into consideration the complex exposure situations and potential confounding factors, they concluded that at exposures <100 ppb such associations between children and adults in homes and schools have generally not been convincing mainly due confounding factors and susceptibility of the findings to chance effects. Furthermore, they did not identify major differences between children, adults, and asthmatic.

This conclusion was upheld in a follow-up review of the same group (Nielsen et al., 2013, key) again concluding that associations between formaldehyde exposure and exacerbation of asthma have not been convincingly demonstrated in children or adults in schools or at home, corresponding to an assessment of Golden (2011, key).

This was also a conclusion of the review of Heinrich (2011, key) who noted that instead consistent environmental risk factors were tobacco smoke, living close to busy roads and in damp homes with visible mould.

Schram-Bijerk et al. (2013, key) came to similar conclusions. They developed a method to estimate the burden of disease (BoD) by the most important indoor air pollutants, namely dampness, carbon monoxide, radon/thoron, formaldehyde and environmental tobacco smoke in The Netherlands. Following a method developed by WHO (Öberg M. Jaakkola MS. Prüss-Üstün A. et al. Second-hand smoke: assessing the environmental burden of disease at national and local levels. Geneva: World Health Organization. 2010) they took into account health outcomes and estimates for exposure levels, % of population exposed, exposure-effect relationships, severity of effect, and duration of disease. The health effect under consideration for formaldehyde was asthma in children. The largest BoD was attributed to environmental tobacco smoke followed by radon and thoron from soils and building materials, dampness and carbon monoxide. According to their estimate, formaldehyde did not contribute to the BoD. They further compared their analysis with BoD estimates from a previous study (EnVIE; de Oliveira Fernandes E, jantunen M, Carrer P. et al. ENVIE-co-ordination action on indoor air quality and health effects. Publishable final activity report. Brussels: European Commission, 2009). The EnVIE estimates were 3 times higher than those of the authors. The most important factors according to EnVIE were combustion products from outdoor sources, bio-aerosols due to dampness and by outdoor sources, volatile organic compounds, radon from soils, pathogens, and carbon monoxide.

Many of the newly identified studies have already been discussed in detail by Nielsen et al. (2013) and the assessments here basically rely on those of Nielsen et al. (2013).

A meta-analysis of McGwin et al. (2010, supporting) with two different models (fixed-effects and random effects model) gave an OR of 1.03 (95 % CI 1.02–1.04) and of 1.17 (95 % CI 1.01–1.36), respectively.

In a cross-sectional study among 2,453 Korean school children respiratory symptoms were obtained by means of a questionnaire. The mean formaldehyde concentration was 28 µg/m³ in class rooms and the outdoor level was 4.3 µg/m³. There was no association between class room FA exposures and wheeze, asthma, but wheeze was significantly associated with reported indoor dampness and mold growth in the home environments (Kim et al. 2011, supporting).

In a cross-sectional study comprising 6,590 children, past-year rhinoconjunctivitis and past year asthma incidences were obtained by questionnaire. Exercise-induced asthma and skin prick test reactions were investigated. A significantly increased odds ratio (OR: 1.19) was observed for past-year rhinoconjunctivitis at formaldehyde exposures exceeding 28.4 µg/m³, but not for past-year asthma (OR: 0.90). Exercise-induced asthma was not correlated with FA exposure. The authors concluded that school air quality may affect rhinitis (formaldehyde) and asthma-related (particulates, acrolein and NO2) morbidity (Annesi-Maesano et al. 2012, supporting).



Justification for classification or non-classification

According to current knowledge and application of the criteria given in Annex I of Regulation (EC) No. 1272/2008, the classification Skin Sens 1A, exceeding the classification given in Regulation (EC) No. 1272/2008, Annex VI, Table 3.1 is required.