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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
year of publication: 1978
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose:
reference to same study
Objective of study:
other: excretion of possible metabolites
Principles of method if other than guideline:
investigation of excretion of possible metabolites in the urine
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Duration and frequency of treatment / exposure:
23 month
Dose / conc.:
9 000 ppm
Remarks:
Doses / Concentrations:
9000 ppm in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on dosing and sampling:
- 2-h urine samples were collected (no further information)
Type:
excretion
Results:
no 3,3'-dichlorobenzidine was detected in the urine
Details on excretion:
- no 3,3'-dichlorobenzidine was detected in the urine
Key result
Test no.:
#1
Toxicokinetic parameters:
other: no 3,3'-dichlorobenzidine was detected in the urine
Conclusions:
The test item is not absorbed after oral application and there is no evidence for a metabolic splitting of the test item to 3,3'-dichlorobenzidine
The test item seems not to be absorbed after oral application and there is no evidence for a metabolic splitting of the test item to 3,3'-dichlorobenzidine.
Executive summary:

Rat were exposed to 9000 ppm test item in diet for two years. Two-hour urine samples were collected after 6 and 23 months from 10 male and female rats and were analysed by thin layer chromatography for the presence of the hypothetical cleavage product 3,3'-dichlorobenzidine. No 3,3'-dichlorobenzidine was detectable in urine of treated and control animals.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Year of publication: 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
other: bioavailability of cleavage products
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity)
Deviations:
yes
Remarks:
e.g. no data on haematology, because the main subject of the study was the investigation of the bioavailability and not the investigation of toxicity after repeated exposure
GLP compliance:
yes
Remarks:
Performance and documentation was on the basis GLP according to the German Chemikaliengesetz, but the test was not under supervision of the quality assurance unit
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: about 3 months
- Weight at study initiation: about 430 g
- Fasting period before study: no data
- Housing: Makrolon cages
- Individual metabolism cages: yes
- Diet: Altromin Pellet-Feed (1324N) or Altromin no Pellet-Feed (1321N) ad libitum
- Water: drinking water ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h/12 h


Route of administration:
other: intratracheal instillation
Vehicle:
other: suspension in isotonic saline containing 1% (w/v) Tween 80
Details on exposure:
- intratracheal instillation of 0.3 ml of suspension
Duration and frequency of treatment / exposure:
once per week for 4 consecutive weeks (day 1, 8, 15, 22 and 29 of the experiment)
Dose / conc.:
0 other: mg/animal/application
Dose / conc.:
10 other: mg/animal/application
Dose / conc.:
20 other: mg/animal/application
No. of animals per sex per dose:
- 12 males per group in the control and test substance group (6 of the animals were sacrificed on day 31 and 6 animals survived till the end of the 4 weeks post exposure period)
- 6 males in the positive control group
Control animals:
yes, concurrent vehicle
Positive control:
5 times 7.31 mg 3,3'-dichlorobenzidine (CAS 91-94-1) or 5 times 5,6 mg Direct Red 46 (CAS 6548-29-4) per animal
Details on study design:
- Dose selection rationale: ascertainment of the maximum tolerable dose in a pretest
Details on dosing and sampling:
BIOAVAILABILITY STUDY
- Tissues and body fluids sampled: urine, faeces, blood, organs (lung, liver, spleen, kidney, urinary bladder, gastro intestinal tract, mamma)
- Time and frequency of sampling of urine and faeces: on day 1, 2, 3, 7, 8, 9, 14, 15, 16, 21, 22, 23, 28, 29, 30 for the animals which were sacrificed on day 31
- Time and frequency of sampling of urine and faeces: on day 28, 29, 30, 35, 42, 49, 56 for the animals which were sacrificed on day 57 at the end of the post exposure period
- Time and frequency of sampling of blood and organs: after sacrifice on day 31 or 57



Statistics:
no data
Type:
absorption
Results:
very limited absorption
Type:
excretion
Results:
3,3'-Dichlorobenzidine was detectable in the urine, and partially in the faeces, likely due to contamination of analytical instruments

 

One animal of the negative control group had to be killed in a moribund condition on day 4. There was no direct correlation to the treatment obvious.

Body-weight development was normal.

The test substance was deposited in the lung: The test substance was detectable in macrophages, in the alveolar and intrabronchiolar region. Lung weights were elevated at the end of the exposure period and still elevated at the end of the post exposure period. Bronchio-alveolar hyperplasia and interstitial fibrosis were detected and persisted till the end of the observation period. Pigment loaded macrophages were also detected in lung associated lymph nodes, which showed increased weight and reactive lymphoide hyperplasia. Effects persisted till the end of the observation time.

3,3'-Dichlorobenzidine was detectable in the hydrolysed urine of treated animals during the exposure period. 3,3'-Dichlorobenzidine excretion via urine was in the range of 3,9 and 4.2 ng/24 hours in the low dose group and about 4.6 ng/24 h on day 2 and 8.8 ng/24 hours on day 30 in the high dose group. 3,3'-Dichlorobenzidine was detectable in two of 30 faeces probes (2.2 ng/24 h and 4.4 ng/24 h). No 3,3'-dichlorobenzidine was verifiable in the faeces and urine during the post exposure period. No 3,3'-dichlorobenzidine was detectable in urine and faeces during the post exposure observation period (measurement on day 42 and 56).

3,3'-Dichlorobenzidine bound to haemoglobin was detectable in 3 out of 5 probes of the low dose group (maximal value: 0.24 ng/g) and in 4 out of 5 probes of the high dose group (maximal value: 6.66 ng/g) at the end of the exposure period. Haemoglobin bound 3,3'-dichlorobenzidine was detectable in 2 out of 5 probes of the high dose group at the end of the post-exposure period (maximal value: 0.13 ng/g).

3,3'-Dichlorobenzidine was also traceable in the urine of several animals of the control group (2 animals sacrificed on day 31 and 5 animals sacrificed on day 57). The authors of the report concluded that this was probably due to a contamination of the collecting containers.

Pigment content of the lungs determined at the end of the exposure period was about 72 -76% of the applied dose. The pigment content of the lungs did not decline during the post exposure period.

The authors calculated that about 0.001 - 0.005% of the theoretically possible 3,3'-dichlorobenzidine was liberated within 24 hours from the applied test substance. Concerning the pigment content of the lung at the end of the exposure period of the high dose group a bioavailability of 0.0012 Mol%/24 hours can be calculated (0.00006 Mol%/24 hours at the end of the post exposure period).

3,3'-dichlorobenzidine treatment:

Animals treated with 3,3'-dichlorobenzidine excreted about 3% of the applied dose via urine (about 194000 ng on day 1 and 226000 ng on day 29) during the first 24 h after dosing. Urinary excretion declined rapidly thereafter: about 10400 ng/24 h during the second and 1030 ng/24 h during the third 24 hours after dosing. The average urinary excretion on the day before the 5th application was 59 ng/24 h. Excretion of 3,3'-dichlorobenzidine via faeces was delayed in comparison to excretion via urine: About 3.6% ( 262 µg; day 1 and 2) or 2.6% (190 µg, day 29 and 30) were excreted within 48 hours after application.

At sacrifice about 1496 ng 3,3'-dichlorobenzidine were bound per g haemoglobin.

Conclusions:
The test substance has a high potential to persist in the lung due to the significantly limited clearance capacity of the lung as a consequence of the particle overload.
Executive summary:

Male Wistar rats were exposed for five times to 10 or 20 mg test item/animal by intratrachel instillation at weekly intervals. Urine and faeces were collected during the exposure and 28 days observation period, blood was collected at the end of the exposure and post observation period. Urine, faeces and haemoglobin were analysed for the presence of 3,3'-dichlorobenzidine after acid hydrolysis, the pigment content of the lungs was determined gravimetrically and the lungs and lung associated lymph nodes were investigated histopathologically. The pigment was deposited in the lung and persisted in the lung during the observation period. 3,3'-Dichlorobenzidine was detectable in the urine, and partially in the faeces and haemoglobin of treated rats during the exposure period. Nevertheless, no proof of bioavailability of the pigment was obtained since 3,3'-Dichlorobenzidine was still detectable in the blood at the end of the post exposure period, but not in the urine and faeces during the post exposure period.

Based on this observation a constant absorption and metabolization of the applied pigment is excluded.

Traces of 3,3'-dichlorobenzidine were also detected in control animals, probably due to impurities of the analytical instruments. Concerning the pigment content of the lung at the end of the exposure period a bioavailability of 0.0012 Mol%/24 hours can be calculated for the high exposure group. At the end of the observation period the bioavailability was only 0.00006 Mol%/24 hours for the high exposure group.

However, since traces of 3,3'-dichlorobenzidine were also found in blood, urine and feces of control animals, it cannot be concluded if the detection of 3,3'-dichlorobenzidine is due to the absorption and biotransformation of the applied pigment or due to the contamination of analytical instruments.

Description of key information

Investigations on the toxicokinetics of diarylide yellow pigments are available for five members of this category:

Pigment Yellow 12, Pigment Yellow 13, Pigment Yellow 17, Pigment Yellow 83, and Pigment Yellow 174. Main focus of these investigations was on the question whether the azo-bonds of these azo-pigments are cleaved under liberation of 3,3'-dichlorobenzidine. After single oral application of the pigments to rat, rabbits, hamsters and monkeys mostly no 3,3'-dichlorobenzidine was detectable in blood and urine, indicating that the test substance is not cleaved under liberation of 3,3'-dichlorobenzidine. No radioactivity was detectable in blood, urine or liver after single oral application of radiolabelled Pigment Yellow 12. Investigations with repeated oral application are somehow contradictory. Whereas the investigations of Zwirner-Baier and Neumann with Pigment Yellow 17 (detection of hemoglobin adducts) indicated that small amounts of the pigment might be cleaved under liberation of 3,3'-dichlorobenzidine, these findings could not be confirmed by Sagelsdorff et al. (1996) using a more sensitive analytical detection method. Therefore, they concluded the the detection of 3,3'-dichlorobenzidine hemoglobin-adducts (Bartsch et al., 2001; Zwirner-Baier and Neumann, 1994) due to insuffucient specificity of the analyticasl method and/or probably due to a contamination of the test material and analytical instruments with the monoazo compound (Sagelsdorff et al., 1996).

After single and repeated inhalation exposure/intratracheal instillation of Pigment Yellow 17 or Pigment Yellow 83 only minimal amounts of 3,3’-dichlorobenzidine, if at all, were detectable in the blood.

Investigations after single dermal application using radiolabelled Pigment Yellow 12 indicate that no test item is absorbed as no radioactivity was detectable in blood, urine or liver and that the test item is excreted via faeces.

In summary, absorption of diarylide yellow pigments of this category after oral, dermal or inhalation application seems to be negligible, if at all. Upon inhalation of a pigment product with a particle size distribution allowing deposition in the lower respiratory tract, uptake and transport of pigments particles by macrophages may occur as for other inert dust particles.

Diarylide yellow pigments of this category are not or only to a negligible extend cleaved under liberation of the carcinogenic compound 3,3’-dichlorobenzidine. Taking into account the negative results obtained with diarylide yellow pigments of this category in investigations on carcinogenicity and genotoxicity in vitro and in vivo it is concluded that this is of no further biological relevance.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Investigations on the toxicokinetics of diarylide yellow pigments have been performed for several members of this category after oral, dermal or inhalative/intratracheal instillation exposure:

Oral exposure:

- single application

After single oral application of radiolabelled Pigment Yellow 12 to rats no radioactivity was detectable in blood, urine and liver during 8 hours following exposure. Radioactivity detected in faeces accounted for the entire applied dose (Decad et al., 1983). After single oral application of 100 mg Pigment Yellow 12/kg bw to male hamsters no metabolites were detectable in urine (Nony et al., 1980). Also, no 3,3'-dichlorobenzidine was detectable in the urine of rats collected for 48 hours after single application of 40 or 400 mg Pigment Yellow 13 or Pigment Yellow 174 per kg bw (CIBA., 1989). Similar results were obtained in rabbits, rats and monkeys which received single oral applications of up to 400 mg Pigment Yellow 13/kg bw: No 3,3'-dichlorobenzidine was detectable in urine collected for 48 hours after dosing (Mondino et al., 1978). No 3,3'-dichlorobenzidine hemoglobin adducts were detectable in rats after single oral application of Pigment Yellow 17 (Zwirner-Baier and Neumann, 1994). In a study which was judged not to be reliable (documentation insufficient for assessment) it was calculated that the amount of 3,3'- dichlorobenzidine detected in the urine of rabbits accounted for 0.05% of the single oral dose of 50 mg Pigment Yellow 13 (Akiyama, 1970).

- repeated application

Formation of 3,3'-dichlorobenzidine (DCB) hemoglobin and liver DNA-adducts was investigated in female rats which were fed for 4 weeks with diet containing 0.2% Pigment Yellow 13 or Pigment Yellow 17 (Sagelsdorff et al., 1996). Neither DCB-hemoglobin adducts nor DCB-DNA-adducts were detectable in rats fed with Pigment Yellow 17 (limits of detection: 0.1 ng/g hemoglobin and 0.08 ng/g DNA). Minimal amounts of DCB-hemoglobin adducts and DCB-DNA adducts were detected in 3/6 rats and 2/3 rats fed with Pigment Yellow 13, respectively. The authors concluded that these adducts are probably due to the contamination of the test item with the monoazo compound and not due to metabolically splitting of the test item into DCB. Zwirner-Baier and Neumann (1994) reported the formation of minimal amounts of 3,3'-dichlorobenzidine hemoglobin adducts after 4 weeks exposure of rats via diet to Pigment Yellow 17 (limit of detection: 6 ng/g hemoglobin). The authors calculated that 0.6% of the total dose of Pigment Yellow 17 was decomposed in the intestine and a corresponding amount of DCB was absorbed. However, this result could not be confirmed by Sagelsdorff et al. (1996), who concluded that the detection of 3,3 -dichlorobenzidine hemoglobin adducts was due to insufficient specificity of the analytical method. No 3,3'- dichlorobenzidine was detectable in the urine of rats after chronic oral treatment with 9000 ppm Pigment Yellow 12 or Pigment Yellow 83 in diet (Leuschner, 1978).

Inhalation exposure:

- single application

Rats were exposed by inhalation to the technically highest administrable concentration of 230 mg Pigment Yellow 17/m3 air for 4 h (nose only; mass-median aerodynamic diameter: 1.0 -1.1 μm). For 14 days after exposure, urine and serum samples ere analysed for 3,3'-dichlorobenzidine. No 3,3'-dichlorobenzidine was detectable, neither in urine nor blood (detection limit 5 ng/ml urine or 10 ng/0.5 ml blood) (Hoechst, 1990; Hofmann and Schmidt, 1993). Minimal 3,3'-dichlorobenzidine hemoglobin adducts which were in the range of the values measured for the control animals were found in rats after single intratracheal application of Pigment Yellow 17 (Zwirner-Baier and Neumann, 1994).

- repeated application

Male Wistar rats were exposed for five times to 10 or 20 mg Pigment Yellow 17 or Pigment Yellow 83/animal by intratracheal instillation at weekly intervals. Urine and faeces were collected during the exposure and 28 days observation period, blood was collected at the end of the exposure and post observation period. Urine, faeces and haemoglobin were analysed for the presence of 3,3'-dichlorobenzidine after acid hydrolysis, the pigment content of the lungs was determined gravimetrically and the lungs and lung associated lymph nodes were investigated histopathologically (Bartsch et al., 2001). Pigment Yellow 17 and Pigment Yellow 83 were deposited in the lung and persisted in the lung during the observation period. No 3,3'- ichlorobenzidine was detectable in the urine, faeces and haemoglobin of Pigment Yellow 17 treated rats during the exposure period. Traces of 3,3'-dichlorobenzidine were detected in control animals, probably due to impurities of the analytical instruments. 3,3'-Dichlorobenzidine was detectable in the urine, and partially in the faeces and haemoglobin of Pigment Yellow 83 treated rats during the exposure period. Nevertheless, no proof of bioavailability of the pigment was obtained, since 3,3'-dichlorobenzidine was still detectable in the blood at the end of the post exposure period, but not in the urine and feces during the post exposure period. Based on this observation a constant absorption and metabolisation of the applied pigment can be excluded. Furthermore, since traces of 3,3'-dichlorobenzidine were also found in blood, urine and feces of control animals, it cannot be concluded if the detection of 3,3'-dichlorobenzidine is due to the absorption and biotranformation of the applied pigment or due to the contamination of analytical instruments. Concerning the pigment content of the lung at the end of the exposure period the authors calculated a bioavailability of 0.0012 Mol%/24 hours for the high exposure group. At the end of the observation period the bioavailability was only 0.00006 Mol%/24 hours for the high exposure group.

Dermal exposure:

- single application

No radioactivity was detectable in blood, urine and liver of male rats after single application of radiolabelled Pigment Yellow 12 to the shaved skin for 24 hours under occlusive conditions. The entire applied dose was detectable on the application site or the patch (Decad et al., 1983).